Methods and products for evaluating an immune response to a therapeutic protein

ABSTRACT

The invention relates to methods and products for the identification of a clinically significant immune response in subjects treated with a therapeutic protein. Aspects of the invention relate to methods and compositions for identifying a clinically significant immune response in patients treated with therapeutic amounts of a VLA4 binding antibody (e.g., natalizumab).

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/330,619, filed Jul. 14, 2014, which is a continuation of U.S.application Ser. No. 13/242,505, filed Sep. 23, 2011, which is acontinuation of U.S. application Ser. No. 11/887,782, filed Mar. 19,2009, now U.S. Pat. No. 8,124,350, which is a national stage filingunder 35 U.S.C. § 371 of international application numberPCT/US2006/012493, filed Apr. 4, 2006, which claims priority under 35U.S.C. § 119(e) from U.S. provisional application Ser. No. 60/668,404,filed Apr. 4, 2005, the entire content of each of which is incorporatedby reference herein.

FIELD OF THE INVENTION

The invention relates to evaluating patients for an immune response to atherapeutic agent, and particularly to a therapeutic protein, forexample a VLA-4 binding antibody (e.g. natalizumab).

BACKGROUND OF THE INVENTION

Biologic therapeutics are currently available for treating diseases anddisorders such as transplant rejection, leukemia, breast cancer,arthritis, multiple sclerosis, and Crohn's disease; and numerousadditional protein-based therapies are in development. Availablebiologics therapeutics include AMEVIVE® (alefacept), ZEVALIN®(ibritumomab tiuxetan), ORTHOCLONE® (muromonab-CD3), ENBREL®(etanercept), REOPRO® (abciximab), RITUXAN® (rituximab), SIMULECT®(basiliximab), REMICADE® (infliximab), SYNAGIS® (palivizumab),HERCEPTIN® (trastuzumab), ZENAPAX® (daclizumab), CAMPATH® (alemtuzumab),MYLOTARG® (gemtuzumab ozogamicin), HUMIRA® (adalimumab), AVONEX®(Interferon beta-1a), and TYSABRI® (natalizumab). Natalizumab is ahumanized monoclonal antibody against α4β1 integrin (VLA-4). Natalizumabbinds to the a4 subunit of α4β1 and α4β1 integrins. Natalizumab isuseful to treat certain inflammatory diseases and conditions includingmultiple sclerosis, Crohn's disease, and rheumatoid arthritis.

Because immune responses to biologic therapeutic agents may haveclinical consequences, immunogenicity assay development and validationis of great importance in the field of biologic therapeutic agents.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for identifying,monitoring, and/or evaluating an immune response to a therapeutic agent,e.g., a therapeutic protein, e.g., a therapeutic antibody. The fact thata patient develops any antibodies to a therapeutic agent (such as atherapeutic protein or therapeutic antibody) may or may not correlatewith a clinical response to the therapeutic agent. Aspects of theinvention are based, in part, on the discovery of an unexpected level ofantibody response that can be used as a threshold for detecting aclinically significant response to the therapeutic agent. In someembodiments, the threshold level is higher than would have beenpredicted using a statistical analysis of patients that have notreceived the therapeutic agent. The clinically significant threshold isgenerally higher than the lowest detectable level of immune response ina patient. For example, the clinically significant threshold level isgenerally at least 2 standard deviations above a negative control level,e.g., above a mean pre-treatment level of an untreated patientpopulation. In some embodiments, the higher threshold levels used inmethods of the invention result in fewer false positives than would beidentified if the threshold level were based on a 5% cutoff (e.g., 1.645standard deviations above the mean) for immune responses observed inpatients that had not received the therapeutic agent. According toaspects of the invention, the presence of a detectable immune responsein a patient sample is not clinically significant unless the immuneresponse reaches at least a predetermined threshold level. The inventionprovides, inter alia, methods of identifying a clinically significantthreshold level of antibody response to a therapeutic agent (e.g., atherapeutic protein, e.g., a therapeutic antibody), and methods ofidentifying patients who have a clinically significant antibody responseto a therapeutic agent. The invention, in part, also provides athreshold level with which to identify clinically meaningful antibodiesin a subject. According to aspects of the invention, an immune responseto a therapeutic agent (e.g., natalizumab) may not be clinicallysignificant (e.g., may not show a significant association with reducedclinical efficacy) unless the magnitude of the immune response reaches athreshold level that can be predetermined (e.g., based on immuneresponses obtained for different patient groups). Surprisingly, themethods described herein do not rely on comparing samples obtained fromeach patient before and after treatment, nor do they rely on identifyingthe mere presence of a detectable immune response to the therapeuticagent. In contrast, methods of the invention, relate to detecting atleast a threshold level of an immune response to a therapeutic agent,where the threshold level may be higher than the lowest detectable levelof immune response, and wherein the positive results from the assay areclinically meaningful, in part, because the assay avoids false positivesthat have no associated clinical significance.

Currently, there is no generally applicable technique or standard fordetecting a clinically significant antibody response to a therapeuticprotein. Different therapeutic proteins may induce different types ofantibodies, and the presence of such antibodies may or may not affectthe safety, pharmacokinetics, and/or efficacy of a therapeutic protein.Current methods of monitoring a patient's response to a therapeuticantibody typically involve comparing levels of serum antibodies beforeand after treatment for each patient identifying the presence of anydetectable immune response, and evaluating the patient to determinewhether the detectable immune response is correlated with any safety,pharmacokinetic, and/or efficacy issues. In contrast, methods of thepresent invention are useful to identify those patients with clinicallysignificant immune responses by providing screening assays for detectingclinically significant threshold levels of response.

According to the invention, a clinically significant immune response toa therapeutic agent is an antibody response that may affect one or moreclinical parameters in a patient, and/or the pharmacokinetics and/orefficacy of the therapeutic agent. Generally, a clinically significantantibody response indicates a diminution of efficacy or lack of efficacyof the therapeutic agent, or an adverse reaction to the therapeuticagent. For example, for multiple sclerosis, a clinically significantantibody response to a therapeutic protein includes one or more of: (a)lack of efficacy or at least 10%, 20%, 30%, 40%, 50%, 60% or morediminution in efficacy of the therapeutic agent to reduce the number,severity or rate of relapse in the patient; (b) lack of efficacy or atleast 10%, 20%, 30%, 40%, 50%, 60% or more diminution in efficacy of thetherapeutic agent to slow progression of disability in the ExpandedDisability Status Scale (EDSS) scale or Multiple Sclerosis FunctionalComposite (MSFC) scale; (c) lack of efficacy or at least 10%, 20%, 30%,40%, 50%, 60% or more diminution in efficacy in reducing the number orvolume of new or newly enlarging T2 hyperintense lesions or attenuatingthe increase in T2 hyperintense lesion volume on brain MRI, (d) lack ofefficacy or at least 10%, 20%, 30%, 40%, 50%, 60% or more diminution inefficacy in reducing the number or volume of gadolinium-enhancinglesions on brain MRI; (e) lack of efficacy or at least 10%, 20%, 30%,40%, 50% 60% or more diminution in efficacy in improving visualfunction; (f) presence of a serious adverse event (e.g.,hypersensitivity reaction, e.g., anaphylaxis). With the exception of(f), such responses are evaluated within a specified period of timeafter administration of the agent, e.g., within 3 months, 6 months, 9months, or at least one year.

In one aspect, the invention provides methods of identifying aclinically significant threshold level of antibody response to atherapeutic agent (e.g., a therapeutic protein, e.g., a therapeuticantibody). The method includes (a) evaluating the level of anti-agentantibodies in a control population of patients who have a disorder(e.g., determining the mean or median level of anti-agent antibodies ina population of at least 2, 3, 5, 10, 20, 30, 50, 100 or more patientswho have a disorder and who have not been treated with a subjecttherapeutic agent for at least 3 months, 6 months or longer); and (b)selecting a threshold level of at least 2 (e.g., 2.5, 3, 4, 5, or 6)standard deviations above the level of anti-agent antibodies in thecontrol population. The presence of at least the threshold level ofanti-agent antibodies in a patient who has been administered thetherapeutic agent (the treated patient) correlates with a clinicallysignificant response in the treated patient. Preferably, the samedetection reagent (e.g., labeled anti-agent antibody) is used toevaluate the treated patient as is used to identify the level ofanti-agent antibodies in the control population. In one embodiment, thetherapeutic agent is a therapeutic antibody, e.g., a humanized 21.6anti-VLA-4 antibody, e.g., natalizumab. In one embodiment, the disorderis multiple sclerosis. In some embodiments, the disorder is aninflammation of the central nervous system (e.g., meningitis,neuromyelitis optica, neurosarcoidosis, CNS vasculitis, encephalitis, ortransverse myelitis, in addition to or instead of multiple sclerosis,),a tissue or organ graft rejection or a graft-versus-host disease, anacute CNS injury (e.g., stroke or spinal cord injury); chronic renaldisease; allergy (e.g., allergic asthma); type 1 diabetes; aninflammatory bowel disorders (e.g., Crohn's disease, or ulcerativecolitis); myasthenia gravis; fibromyalgia; an arthritic disorder (e.g.,rheumatoid arthritis or psoriatic arthritis); an inflammatory/immuneskin disorder (e.g., psoriasis, vitiligo, dermatitis, or lichen planus);systemic lupus erythematosus; Sjogren's Syndrome; a hematological cancer(e.g., multiple myeloma, leukemia, or lymphoma); a solid cancer such asa sarcoma or a carcinoma (e.g., of the lung, breast, prostate, orbrain); or a fibrotic disorder (e.g., pulmonary fibrosis, myelofibrosis,liver cirrhosis, mesangial proliferative glomerulonephritis, crescenticglomerulonephritis, diabetic nephropathy, or renal interstitialfibrosis). In some embodiments, the disorder is a disease that involvesmodulation of an α4β1 and/or α4β7 subunit.

In another aspect, the invention provides methods of identifying apatient who has a clinically significant antibody response to atherapeutic protein, e.g., a therapeutic antibody. The method includesidentifying, in a biological sample obtained from a subject who has adisorder and who has been administered the therapeutic protein, thepresence of a threshold level of one or more antibodies thatspecifically bind to the therapeutic protein, wherein the thresholdlevel is at least 2 (e.g., 2.5, 3, 4, 5, or 6) standard deviations abovethe level of antibodies that specifically bind to the therapeuticprotein in a control population (e.g., a population of patients who havethe disorder but have not been administered the therapeutic proteinwithin the last 3 months, 6 months or more). In one embodiment, thetherapeutic protein is a therapeutic antibody, e.g., a humanized 21.6(also referred to as AN100226) anti-VLA-4 antibody, e.g., natalizumab.In one embodiment, the disorder is multiple sclerosis. In someembodiments, the disorder is rheumatoid arthritis. In certainembodiments, the disorder is Crohn's disease. In one embodiment, themethod further includes modifying the treatment regimen of a patient whois thus identified as having a clinically significant antibody responseto a therapeutic protein.

In one aspect, the invention provides methods and compositions foridentifying in a biological sample obtained from a subject the presenceof a clinically significant level of one or more antibodies thatspecifically bind to a therapeutic VLA-4 binding antibody that wasadministered to the subject. Aspects of the invention include the use ofELISA assays for the detection of levels of induced antibodies that areindicative of a clinically significant immune response in a subject tothe administration of a therapeutic VLA-4 binding antibody. In oneembodiment, the invention provides methods and kits for identifyingclinically significant levels of anti-natalizumab antibodies that areindicative of an immune response to natalizumab in a subject that hasreceived at least one dose of natalizumab

In one aspect, the invention provides methods for evaluating and/ormodifying a therapeutic regimen based on a subject's immune response toa VLA-4 binding antibody.

According to one aspect of the invention, methods of detecting aclinically significant immune response to a VLA-4 binding antibody in asubject are provided. The methods include determining whether abiological sample from a subject that has been administered a VLA-4binding antibody contains a clinically significant threshold level of asoluble antibody that binds to the VLA-4 binding antibody, wherein thepresence of at least the threshold level of the soluble antibody isindicative of a clinically significant immune response to the VLA-4binding antibody. In some embodiments, a clinically significant immuneresponse to the VLA-4 binding antibody is indicated by the presence ofat least the threshold level of soluble antibody to the VLA-4 bindingantibody in at least two biological samples taken from the subject atdifferent time points. In certain embodiments, the time points areseparated by at least one month. In some embodiments, at least thethreshold level of soluble antibody that binds to the VLA-4 bindingantibody is present in two biological samples taken from the subject attwo consecutive time points. In some embodiments, a level of solubleantibody that binds to the VLA-4 binding antibody is determined by:determining a level of soluble binding activity to the VLA-4 bindingantibody in a first aliquot of the biological sample; and determiningwhether the soluble binding activity is specific for the VLA-4 bindingantibody. In certain embodiments, the specificity of the soluble bindingactivity is determined in a second aliquot of the biological sample. Insome embodiments, a level of soluble antibody that binds to the VLA-4binding antibody in the biological sample is determined by comparinglevels of binding activity to a labeled VLA-4 binding antibody measuredin the presence of two or more different amounts of unlabeled VLA-4binding antibody (e.g., levels measured in the presence of no unlabeledVLA-4 binding antibody may be compared to levels measured in thepresence of a competing amount of unlabeled VLA-4 binding antibody). Incertain embodiments, a level of soluble antibody that binds to the VLA-4binding antibody in the biological sample is determined by comparinglevels of binding activity to an immobilized VLA-4 binding antibodymeasured in the presence of two or more different amounts of solubleVLA-4 binding antibody (e.g., levels measured in the presence of nosoluble VLA-4 binding antibody may be compared to levels measured in thepresence of a competing amount of soluble VLA-4 binding antibody). Insome embodiments, a first level of binding activity to a labeled VLA-4binding antibody measured in the presence of a first amount of unlabeledVLA-4 is compared to a second level of binding activity to a labeledVLA-4 binding antibody measured in the presence of a second amount ofunlabeled VLA-4 binding antibody. In some embodiments, the first andsecond levels of binding activity are determined in first and secondaliquots of the biological sample. In certain embodiments, the amount ofsoluble antibody to the VLA-4 binding antibody is determined using abridging ELISA assay. In some embodiments, a first level of bindingactivity to the VLA-4 binding antibody is determined in a firstimmunoassay for a first aliquot of the biological sample, and a secondlevel of binding activity to the VLA-4 binding antibody is determined ina second immunoassay for a second aliquot of the biological sample,wherein the second immunoassay is spiked with a greater amount ofunlabeled soluble VLA-4 binding antibody than the first immunoassay, andwherein the presence in the biological sample of at least a thresholdlevel of soluble antibody to the VLA-4 binding antibody is indicated ifthe first level of binding activity is greater than a reference leveland the second level of binding activity is less than a predeterminedpercentage of the first level of binding activity. In certainembodiments, the reference level is a level of binding activity measuredfor a reference amount of soluble antibody to the VLA-4 bindingantibody. In some embodiments, the reference amount is about 500 ng/ml(e.g., in a serum sample) of a soluble antibody to the VLA-4 bindingantibody. For example, the reference amount may be between about 400ng/ml and about 600 ng/ml (e.g., about 400 ng/ml, about 425 ng/ml, about450 ng/ml, about 475 ng/ml, about 500 ng/ml, about 525 ng/ml, about 550ng/ml, about 575 ng/ml, or about 600 ng/ml). It should be appreciatedthat the reference level of binding activity that corresponds to thereference amount may be measured in a diluted sample (for example, asample that corresponds to a 10 fold dilution and contains from about 40ng/ml to about 60 ng/ml, e.g., about 50 ng/ml, of a soluble antibodythat to the VLA-4 binding antibody). A reference level of bindingactivity in an assay may be provided by any predetermined amount ofsoluble antibody to the VLA-4 binding antibody corresponding to anappropriate dilution of the reference amount. In certain embodiments,the VLA-4 binding antibody is a humanized murine monoclonal antibody toVLA-4. In some embodiments, the VLA-4 binding antibody is a humanizedform of murine antibody mAb 21.6, (e.g., AN100226). In some embodiments,the VLA-4 binding antibody is natalizumab. In some embodiments, thesoluble antibody to the VLA-4 binding antibody is a reference antibodythat binds to natalizumab with high affinity. In some embodiments, thereference antibody blocks the interaction between natalizumab and VLA-4.In certain embodiments, the first and second immunoassays are bridgingELISA assays. In some embodiments, the first and second assays comprisean immobilized unlabeled VLA-4 binding antibody and a soluble labeledVLA-4 binding antibody, wherein the soluble labeled VLA-4 bindingantibody is labeled with an enzyme, a fluorescent marker, a biotinmarker (e.g., the VLA-4 binding antibody may be biotinylated), or aradioactive marker. In some embodiments, the first and secondimmunoassays are conducted in parallel reaction volumes on a singlereaction substrate. In some embodiments, the biological sample is aserum sample. In certain embodiments, the subject is a human patient. Insome embodiments, the patient has multiple sclerosis. In someembodiments, the patient has rheumatoid arthritis. In certainembodiments, the patient has Crohn's disease. In some embodiments, thetime points of at least two or more biological samples obtained thesubject are separated by at least 15 days, 30 days, 45 days, 60 days, 90days, or more. In certain embodiments, the method also includesselecting a therapeutic regimen for the subject if a clinicallysignificant threshold level of a soluble antibody that binds to theVLA-4 binding antibody is detected in at least two biological samplesobtained from the subject. In some embodiments, selecting a therapeuticregimen includes evaluating a current therapy of the subject,determining a new therapy for the subject, modifying a current therapyof the subject, or stopping a current therapy of the subject. In someembodiments, a current therapy includes administering the VLA-4 bindingantibody to the subject.

According to another aspect of the invention, methods of selecting atherapeutic regimen for a subject are provided. The methods includeassaying a subject who has been administered a VLA-4 binding antibodyfor the presence of a positive immune response to the VLA-4 bindingantibody at first and second time points, selecting a therapeuticregimen for the subject based on the assay results at the first andsecond time points, wherein the presence of a positive immune responseat a point in time is indicated by the presence of at least a clinicallysignificant threshold amount of binding activity in a biological sampleobtained from the subject at the point in time. In some embodiments, thefirst and second time points are separated by a clinically significanttime period. In certain embodiments, the clinically significant timeperiod is at least 30 days. In some embodiments, VLA-4 binding antibodytherapy is continued if a negative immune response is detected at thesecond time point. In some embodiments, a therapy other than VLA-4binding antibody therapy is selected if a positive immune response isdetected at both the first and second time points. In some embodiments,the subject has multiple sclerosis. In certain embodiments, the subjecthas Crohn's disease. In some embodiments, the subject has rheumatoidarthritis.

According to yet another aspect of the invention, methods of selecting atherapeutic regimen for a subject are provided. The methods includedetecting the presence of a clinically significant immune response to aVLA-4 binding antibody in at least two biological samples obtained froma subject, wherein the subject has been administered a VLA-4 bindingantibody and the at least two biological samples are obtained from thesubject at times separated by at least a clinically significant timeinterval, and selecting a therapeutic regimen based on the detection ofa clinically significant immune response to the VLA-4 antibody in thesubject at the times when the at least two biological samples areobtained from the subject. In some embodiments, the clinicallysignificant interval separating the times at which the samples areobtained from the subject is at least 15 days, 30 days, 45 days, 60days, 90 days, or longer. In certain embodiments, selecting atherapeutic regimen includes evaluating a current therapy of thesubject, determining a new therapy for the subject, modifying a currenttherapy of the subject, or stopping a current therapy of the subject. Insome embodiments, a current therapy includes administering the VLA-4antibody to the subject. In some embodiments, detecting the presence ofa clinically significant immune response to a VLA-4 binding antibodycomprises, determining whether a biological sample obtained from asubject that has been administered a VLA-4 binding antibody contains athreshold level of a soluble antibody that binds to the VLA-4 bindingantibody, wherein the presence of at least the threshold level of thesoluble antibody is indicative of a clinically significant immuneresponse to the VLA-4 binding antibody. In certain embodiments, at leastthe threshold level of soluble antibody that binds to the VLA-4 bindingantibody is present in two biological samples taken from the subject attwo consecutive time points. In some embodiments, a level of solubleantibody that binds to the VLA-4 binding antibody is determined by:determining a level of soluble binding activity to the VLA-4 bindingantibody in a first aliquot of the biological sample and determiningwhether the soluble binding activity is specific for the VLA-4 bindingantibody. In some embodiments, the specificity of the soluble bindingactivity is determined in a second aliquot of the same biologicalsample. In certain embodiments, a level of soluble antibody that bindsto the VLA-4 binding antibody in the biological sample is determined bycomparing levels of binding activity to a labeled VLA-4 binding antibodymeasured in the presence of two or more different amounts of unlabeledVLA-4 binding antibody (e.g., levels measured in the presence of nounlabeled VLA-4 binding antibody may be compared to levels measured inthe presence of a competing amount of unlabeled VLA-4 binding antibody).In some embodiments, a level of soluble antibody that binds to the VLA-4binding antibody in the biological sample is determined by comparinglevels of binding activity to an immobilized VLA-4 binding antibodymeasured in the presence of two or more different amounts of solubleVLA-4 binding antibody (e.g., levels measured in the presence of nosoluble VLA-4 binding antibody may be compared to levels measured in thepresence of a competing amount of soluble VLA-4 binding antibody). Insome embodiments, a first level of binding activity to a labeled VLA-4binding antibody measured in the presence of a first amount of unlabeledVLA-4 is compared to a second level of binding activity to a labeledVLA-4 binding antibody measured in the presence of a second amount ofunlabeled VLA-4 binding antibody. In certain embodiments, the first andsecond levels of binding activity are determined in first and secondaliquots of the same biological sample. In some embodiments, the amountof soluble antibody to the VLA-4 binding antibody is determined using abridging ELISA assay. In some embodiments, a first level of bindingactivity to the VLA-4 binding antibody is determined in a firstimmunoassay for a first aliquot of the biological sample, and a secondlevel of binding activity to the VLA-4 binding antibody is determined ina second immunoassay for a second aliquot of the biological sample,wherein the second immunoassay is spiked with a greater amount ofunlabeled soluble VLA-4 binding antibody than the first immunoassay, andwherein the presence in the biological sample of at least a thresholdlevel of soluble antibody to the VLA-4 binding antibody is indicated ifthe first level of binding activity is greater than a reference leveland the second level of binding activity is less than a predeterminedpercentage of the first level of binding activity. In some embodiments,the reference level is a level of binding activity measured for areference amount of soluble antibody to the VLA-4 binding antibody. Incertain embodiments, the reference amount is about 500 ng. In someembodiments, the VLA-4 binding antibody is a humanized murine monoclonalantibody to VLA-4. In some embodiments, the VLA-4 binding antibody is ahumanized form of murine antibody mAb 21.6. In some embodiments, theVLA-4 binding antibody is natalizumab. In certain embodiments, the firstand second immunoassays are bridging ELISA assays. In some embodiments,the first and second assays comprise an immobilized unlabeled VLA-4binding antibody and a soluble labeled VLA-4 binding antibody, whereinthe soluble labeled VLA-4 binding antibody is labeled with an enzyme, afluorescent marker, a biotin marker (e.g., the VLA-4 binding antibodymay be biotinylated), or a radioactive marker. In some embodiments, thefirst and second immunoassays are conducted in parallel reaction volumeson a single reaction substrate (e.g., in separate wells of a multi-wellplate). In certain embodiments, the biological sample is a serum sample.In some embodiments, the subject is a human patient. In someembodiments, the patient has multiple sclerosis. In some embodiments,the patient has rheumatoid arthritis. In certain embodiments, thepatient has Crohn's disease.

According to another aspect of the invention, methods of identifying aclinically significant threshold level of antibody response to a proteintherapeutic agent for a patient who has a disorder are provided. Themethods includes (a) evaluating the level of anti-agent antibodies in acontrol population of patients who have the disorder and who have notbeen treated with the agent and (b) selecting a level at least 2standard deviations above the level of anti-agent antibodies in thecontrol population as a clinically significant threshold level ofantibody response for patients who have the disorder and are treatedwith the agent. In some embodiments, the protein therapeutic agent is atherapeutic antibody or antigen-binding fragment thereof.

According to yet another aspect of the invention, methods of identifyinga patient who has a clinically significant antibody response to atherapeutic protein are provided. The methods include assaying, in abiological sample obtained from a patient who has a disorder and who hasbeen administered the therapeutic protein, for the presence of athreshold level of antibodies that specifically bind to the therapeuticprotein, wherein the threshold level is at least 2 standard deviationsabove the level of antibodies that specifically bind to the therapeuticprotein in a control untreated population of patients who have thedisorder. In some embodiments, the therapeutic protein is a therapeuticantibody or antigen-binding fragment thereof. Accordingly aspects of theinvention provide methods of detecting a clinically significant immuneresponses to a VLA-4 binding antibody in a subject, by determiningwhether a biological sample from a subject that has been administered aVLA-4 binding antibody contains a threshold level of a soluble antibodythat binds to the VLA-4 binding antibody, wherein the presence of atleast the threshold level of the soluble antibody is indicative of aclinically significant immune response to the VLA-4 binding antibody.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the interference of free natalizumab in animmunoassay.

FIG. 2 illustrates the interference of free natalizumab in a blockingassay.

FIG. 3 shows a probability of a clinically significant immune responseas a function of time after an initial administration of natalizumab.

FIG. 4 illustrates an overall effect of positive immune responses onrelapse rates. P=Placebo, n=315. AN=Antibody Negative, n=569.TP=Transient Positive, n=19. PP=Persistent Positive, n=37.

FIGS. 5A-5D illustrate the effect of positive immune responses onrelapse rates over particular time intervals after an initialadministration of natalizumab (FIG. 5A: 0-3 months, FIG. 5B: 3-6 months,FIG. 5C: 6-9 months, and FIG. 5D: 9-12 months). P=Placebo, n=315.AN=Antibody Negative, n=569. TP=Transient Positive, n=19. PP=PersistentPositive, n=37.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in part, to methods, compositions, andkits for detecting and monitoring an immune response to a therapeuticprotein (e.g., a therapeutic antibody) that is administered to asubject. In one aspect, the invention provides methods for identifyingpatients with a clinically significant immune response to a therapeuticantibody. According to the invention, the presence of a detectableimmune response is not clinically significant unless the immune responsereaches a clinically significant threshold level. For example, inclinical studies of natalizumab, a clinically significant thresholdlevel of immune response was surprisingly more than 1.645 standarddeviations (e.g., at more than about 2 standard deviations) above acontrol level of immune response observed for subjects that have notreceived the therapeutic agent.

Certain aspects of the invention relate to methods for detecting aclinically significant immune response against a therapeutic VLA-4binding antibody that is administered to a subject. Aspects of theinvention are particularly useful for detecting and monitoring immuneresponses in a subject who has received at least one dose (e.g., onetherapeutic dose) of a VLA-4 binding antibody. Aspects of the inventioninclude identifying and/or monitoring a subject with a clinicallysignificant immune response to a therapeutic VLA-4 binding antibody,evaluating the immune response, and/or determining an appropriateclinical treatment (e.g., a particular therapeutic regimen) based on thenature and/or extent of the immune response. Information about asubject's response to the administration of a VLA-4 binding antibody maybe used to adjust, design, and/or optimize a therapeutic regimen for thesubject. Accordingly, one aspect of the invention relates to identifyinga subject who has a clinically significant immune response to atherapeutic VLA-4 binding antibody. Another aspect of the inventionrelates to monitoring a subject's immune response to a therapeutic VLA-4binding antibody. A further aspect of the invention relates todetermining appropriate therapeutic strategies to treat certain diseases(e.g., multiple sclerosis, Crohn's disease, or rheumatoid arthritis,etc.) based on a subject's immune response to a therapeutic VLA-4binding antibody.

VLA-4 binding antibodies may be used to treat a number of diseases anddisorders associated with inflammation. Such disorders include, e.g.,inflammation of the central nervous system (e.g., in addition tomultiple sclerosis, meningitis, neuromyelitis optica, neurosarcoidosis,CNS vasculitis, encephalitis, and transverse myelitis), tissue or organgraft rejection or graft-versus-host disease, acute CNS injury, e.g.,stroke or spinal cord injury; chronic renal disease; allergy, e.g.,allergic asthma; type 1 diabetes; inflammatory bowel disorders, e.g.,Crohn's disease, ulcerative colitis; myasthenia gravis; fibromyalgia;arthritic disorders, e.g., rheumatoid arthritis, psoriatic arthritis;inflammatory/immune skin disorders, e.g., psoriasis, vitiligo,dermatitis, lichen planus; systemic lupus erythematosus; Sjogren'sSyndrome; hematological cancers, e.g., multiple myeloma, leukemia,lymphoma; solid cancers, e.g., sarcomas or carcinomas, e.g., of thelung, breast, prostate, brain; and fibrotic disorders, e.g., pulmonaryfibrosis, myelofibrosis, liver cirrhosis, mesangial proliferativeglomerulonephritis, crescentic glomerulonephritis, diabetic nephropathy,and renal interstitial fibrosis. In some embodiments, the disorder is adisease effected by modulation of an α4β1 or/an α4β7 subunit.

In one embodiment, a VLA-4 binding antibody is a humanized version ofmurine mAb 21.6, e.g., natalizumab. Accordingly, aspects of theinvention relate to evaluating a subject's response to natalizumab anddetermining appropriate treatments for multiple sclerosis and otherinflammatory conditions or diseases that can be treated withnatalizumab.

The invention relates in part to identifying an immune response to aVLA-4 binding antibody (e.g., a humanized version of murine mAb 21.6,such as natalizumab, or AN100226) in a subject, and determining whetherthe response is clinically significant.

As used herein, “identifying” a subject with an immune response meansdetecting or diagnosing the presence of an immune response in a subject.Accordingly, identifying a subject with a clinically significant immuneresponse means detecting or diagnosing the presence of a clinicallysignificant immune response in a subject.

As used herein, a “clinically significant threshold” for an antibodyresponse to a therapeutic protein is at least 2 standard deviationsabove a control reference level. In one embodiment, the threshold levelfor a clinically significant immune response to a therapeutic proteinmay be between 3 and 6 (e.g., about 4 or 5) standard deviations above acontrol level. The control level may be a mean or median level ofbinding activity that is present in a patient population (e.g., apopulation of subjects with a disease or condition such as multiplesclerosis, Crohn's disease, or rheumatoid arthritis) before exposure tothe therapeutic protein. In one embodiment, a clinically significantthreshold for anti-natalizumab antibodies is 500 ng/ml of patient sera(e.g., a 50 ng/ml threshold in an assay of 10-fold diluted serum).

As used herein, an immune response is an immunogenic response to atherapeutic protein characterized by increased levels in the subject ofone or more antibodies that bind the protein. Thus, an immune responsemay be characterized by the induction of increased levels of solubleantibodies that recognize (e.g., specifically recognize) and bind to theprotein, e.g., a VLA-4 binding antibody (e.g. natalizumab). A typicalimmune response is polyclonal and may include antibodies with differentaffinities (and therefore different degrees of specificity) for thetherapeutic protein. Accordingly, methods of the invention may involvedetecting the presence in a subject of one or more induced antibodiesthat bind to a therapeutic protein (e.g., a VLA-4 binding antibody) thatwas administered to the subject. In some embodiments, the inducedantibodies may be detected as soluble antibodies that are present in abiological sample (e.g., a serum sample).

Aspects of the invention relate to assays for detecting a clinicallysignificant threshold level of binding activity in a biological sampleobtained from a patient. The threshold level represents a level belowwhich any detectable binding activity is considered not to be clinicallysignificant. As used herein, binding activity refers to the detectedamount of binding to a therapeutic protein in a biological sample. Asdescribed herein, the presence of binding activity in a biologicalsample may reflect a polyclonal response to the administration of atherapeutic protein. Accordingly, the amount of binding may reflect anaggregate of binding by different antibodies with different affinitiesfor the protein. In certain embodiments, the binding activity is furtheranalyzed to determine with greater confidence whether the level ofbinding is due to the presence of specific antibodies against thetherapeutic protein or due to other factors such as rheumatoid factors.The specificity of a binding activity may be evaluated in competitionassays as described herein.

In one aspect of the invention, a subject is identified as a positive(i.e., as having a clinically significant immune response to atherapeutic protein) only if one or more samples obtained from thesubject test positive in an assay of the invention. A positive testresult is determined when a sample obtained from a subject contains atleast a clinically significant threshold level of binding activity forthe therapeutic protein, e.g., VLA-4 binding antibody. Surprisingly, thepresence of any detectable immune response to a therapeutic antibody isnot clinically significant. According to the invention, methods based onscreening patients to detect any level of immune response to atherapeutic antibody identify many false positive patients, resulting inunnecessary additional clinical monitoring and potential anxiety forpatients who do not have a clinically significant immune response. Forexample, an excessive number of false positives are detected whenpatients are identified as positive based on an immune response to atherapeutic antibody that is greater than 1.645 standard deviationsabove a mean level of binding activity present in subjects that have notreceived the therapeutic antibody. According to the invention, thetheoretical 5% false positive rate using a 1.645 standard deviationcut-off is an underestimate of the number of false positives, becausethe 5% represents the rate of false-detection of any immune response andnot the rate of false-positives for a clinically significant immuneresponse. Surprisingly, by raising the cut-off level (the level belowwhich a response is considered to be negative) to higher than 1.645standard deviations above a control reference level, the number of falsepositives is reduced without affecting the identification of subjectswith clinically significant immune responses. It should be appreciatedthat the threshold should be set at a level that results in acceptabledetection rates of patients with a clinically significant immuneresponse. Therefore, even though the clinically significant thresholdshould be set at more than 1.645 standard deviations above a pre-immunereference level, the threshold should not be set so high as to reducethe detection efficiency of actual positives.

In one aspect of the invention, a subject's immune response may beclassified as negative if samples obtained from the subject do not testpositive in an assay of the invention, e.g., they do not reach theclinically significant threshold level of antibody response. Incontrast, if a subject is identified as positive based on a positivelevel (a level at or above a clinically significant threshold level) ofbinding activity in a single assay, the patient may be either a“transient” or a “persistent” positive. A transient positive is apatient who has a positive immune response to the therapeutic antibodyfor a specified period of time after which the patient becomes negative.In contrast, a persistent positive is a patient who is positive forclinically significant levels of immune response for greater than aspecified period of time. It should be appreciated that transient andpersistent are relative terms. Accordingly, a patient may be classifiedinitially as persistent if the patient tests positive for an immuneresponse at two or more time points (e.g., at 3, 4, 5, 6, 7, 8, 9, 10 ormore time points) separated by clinically significant time intervals.However, the patient subsequently may be reclassified as transient ifthe patient tests negative for an immune response in a subsequent assay.Clinically significant time intervals may be at least one week, onemonth, one year, or longer. For example, the threshold time interval maybe between 30 and 180 days, about 60 days, about 42 days, etc. Thepresence of a transient immune response may be indicative of a transientreduction in therapeutic efficacy. The presence of a persistent immuneresponse may be indicative of a persistently reduced therapeuticefficacy. Accordingly, the presence of a transient or persistent immuneresponse may be clinically relevant and may affect the nature of atherapeutic regimen in a subject that is identified as transientlypositive or persistently positive. A persistent immune response maynecessitate a modification of the subject's therapeutic regimen.

As used herein, the term “therapeutic regimen” means a course oftreatment for a subject. A therapeutic regimen may includeadministration of pharmaceutical agent(s) and/or application of atherapy. The selection of a regimen may include selection of doseamount, dose timing, dose frequency, duration of treatment, combinationtherapies with one or more pharmaceutical agents or therapies, and anyother aspects of treatment decision making that are used by those ofskill in the medical and therapeutic arts. A therapeutic regimen alsomay include the use of therapies such as procedures or devices that areadministered to or used on a subject for the prevention or treatment ofa disease or disorder. Examples of therapeutic procedures, although notintended to be limiting, include the use of medical devices or surgery.Accordingly, determining or altering a VLA-4 binding antibodytherapeutic regimen may involve determining or altering the dose amountof therapeutic VLA-4 binding antibody that is administered to a subject,the frequency of administration, the route of administration, theduration of the treatment (e.g., the number of doses that areadministered), whether or not to combine a VLA-4 binding antibodytreatment with one or more additional treatments, whether to discontinuea VLA-4 binding antibody treatment, whether to use a different VLA-4binding antibody, and/or whether to use a combination of VLA-4 bindingantibodies, etc. In one embodiment, the invention may involvedetermining whether to use a therapeutic alternative to a VLA-4 bindingantibody, e.g., whether to use beta interferon.

Aspects of the invention relate to detecting and/or monitoring an immuneresponse to a VLA-4 binding antibody in a human (e.g., a human patient).Accordingly, as used herein, a subject may be a human subject. A subjectmay be a human patient that has an inflammatory disease or condition. Asubject may be a patient that has received at least one dose of a VLA-4binding antibody (e.g., natalizumab). A subject may be a patient that isbeing (or was) treated chronically with a VLA-4 binding antibody (e.g.,natalizumab). A subject may be a patient that is being (or was) treatedrepeatedly with a VLA-4 binding antibody (e.g., natalizumab). As usedherein, a chronic treatment may involve administering a VLA-4 bindingantibody over an extended period of time (e.g., to control or managesymptoms of an inflammatory disease or condition during the timeperiod). In contrast, a repeated treatment may involve repeating acourse of treatment (e.g., a period of administration) with a VLA-4binding antibody when necessary (e.g., to treat symptoms of aninflammatory disease when they worsen or “flare up”). In one embodiment,a patient is considered to be undergoing a repeated treatment when thesubject is re-treated with a therapeutic VLA-4 binding antibody for thefirst time. It will be understood by those of ordinary skill in the artthat a subject may also undergo or have undergone treatments withtherapies or procedures in combination with or separate from treatmentwith a VLA-4 binding antibody. It should be appreciated that aspects ofthe invention are not limited to human subjects. Accordingly, a subjectmay be a non-human primate, cow, horse, pig, sheep, goat, dog, cat,rodent, or other non-human subject.

Identifying and Monitoring an Immune Response

In one aspect, the invention involves identifying and/or monitoring animmune response to a VLA-4 binding antibody in a subject. In certainembodiments, the identification and/or monitoring is performed byassaying a biological sample obtained from the subject, preferablyblood, for the presence of induced antibodies that bind to theadministered VLA-4 binding antibody as described herein.

In one aspect of the invention, a qualitative assay is performed on abiological sample obtained from a subject, and the presence of an immuneresponse is identified if the biological sample contains antibodiesagainst the VLA-4 binding antibody in an amount greater than a thresholdamount. In one embodiment, a threshold amount is an amount above whichan immune response is identified as being clinically relevant, e.g., thethreshold level is determined as described herein. A clinically relevantimmune response may have clinical implications, e.g., it indicates thatthe subject should be evaluated to determine whether the dosage of theadministered VLA-4 binding antibody should be modified, to determinewhether other physiological parameters of the patient should bemonitored, to determine whether a further assay for an immune responseshould be performed, or to determine whether any alternative oradditional steps should be taken to treat or monitor the subject, etc. Aclinically relevant immune response may be evaluated along with one ormore other factors. It should be appreciated that the identification ofa clinically relevant immune response does not, by itself, require thata change be made to the subject's therapy or treatment regimen.

In another aspect of the invention, a quantitative assay may beperformed on a biological sample to quantify the amount of antibodies(e.g., the antibody titer) against a VLA-4 binding antibody that wasadministered to a subject. Quantitative results also may be analyzed todetermine whether an immune response is above a clinically significantthreshold level.

According to the invention, an immune response against a VLA-4 bindingantibody (e.g., natalizumab) may be assessed in a subject over time byperforming assays on samples obtained at different time points from thesubject. The multiple-assessment strategy permits monitoring of asubject's immune response to the VLA-4 binding antibody and may allowthe therapeutic VLA-4 binding antibody regimen to be individuallytailored to the subject's therapeutic needs. For example, a sample maybe obtained from a subject, tested for an immune response to the VLA-4binding antibody that has been administered to the subject, and at asecond, subsequent time, another sample may be obtained from the subjectand similarly tested. Detection and confirmation of the presence of anantibody response in a subject's samples over time by sequentialdeterminations at predetermined time intervals permits monitoring of animmune response to a therapeutic VLA-4 binding antibody treatment. Thedetection and monitoring of an immune response to an administered VLA-4binding antibody also allows adjustment in the overall treatment of thesubject, for example by adjusting (e.g., modifying or suspending) theVLA-4 binding antibody treatment and/or by adjusting additionaltherapies (e.g., therapies that modulate the immune response of thesubject).

The selection or adjustment of a therapeutic regimen may be based on adetermination of a clinically significant immune response to a VLA-4binding antibody in at least two biological samples obtained atdifferent times from a subject who has been administered a VLA-4 bindingantibody. The determination of a subject's clinically significant immuneresponse to the VLA-4 binding antibody may indicate that initiating,continuing, adjusting, or stopping administration of a specificpharmaceutical agent and/or therapy to the subject would be beneficial.For example, the determination of a clinically significant immuneresponse to a VLA-4 binding antibody in at least two biological samplesobtained from a subject may be the basis for altering the dose of apharmaceutical agent that is administered to the subject as part of acurrent therapeutic regimen. The treatment may be changed to includeadditional pharmaceutical agents or therapies or to lower or raise thedose of a currently administered agent or therapy. For example, theidentification of an immune response to a VLA-4 binding antibody in asubject may suggest initiating or continuing a treatment with animmunosuppressive pharmaceutical agent, etc. In some embodiments, aninitial therapeutic regimen may be selected based on the determinationof an initial immune response to a VLA-4 binding antibody in a singlebiological sample obtained from a subject who has been treated with aVLA-4 binding antibody. Following the selection and administration of aselected therapeutic regimen, a subsequent determination of an immuneresponse to a VLA-4 binding antibody in one or more subsequentbiological samples obtained from the subject may be made and may providea basis for adjusting the therapeutic regimen.

The determination of a clinically significant immune response to a VLA-4binding antibody in two or more biological samples obtained from asubject at different time points can be compared to evaluate or measurethe onset, progression, or regression of an immune response in thesubject to the VLA-4 binding antibody therapy. Onset of an immuneresponse to a VLA-4 binding antibody in a subject may be characterizedby increased level(s) of at least one antibody that binds to a VLA-4binding antibody, and may be accompanied by the onset of one or morephysiological changes or symptoms in the subject. Progression of animmune response to a therapeutic VLA-4 binding antibody may becharacterized by a further increase in the level of the at least oneantibody that binds to the therapeutic VLA-4 binding antibody. However,the progression of an immune response may involve an increase in thelevel(s) of at least one additional antibody, and/or a decrease in thelevel of at least one of the antibodies that increased with the onset ofthe immune response. For example, an initial immune response may bepredominantly an IgM response. As the immune response progresses, thepredominant antibodies may switch from IgM to IgG antibodies.Progression of an immune response also may be accompanied by aprogression (e.g., an increase, decrease, or modification) of one ormore of the initial physiological changes or symptoms or the onset ofone or more additional physiological changes or symptoms. Regression ofan immune response in a subject to a therapeutic VLA-4 binding antibodymay be characterized by a decrease in the level(s) of one or moreantibodies that bind to the therapeutic VLA-4 binding antibody. Theregression of an immune response also may be accompanied by a decreaseof certain physiological changes or symptoms. However, it should beappreciated that onset, progression, and/or regression of an immuneresponse to a therapeutic VLA-4 binding antibody may be clinicallyasymptomatic, other than the detectable changes in antibody levels.

Progression and regression of a clinically significant immune responseto a VLA-4 binding antibody may generally be indicated by the increaseor decrease, respectively, of the level of an antibody that binds aVLA-4 binding antibody in a subject's samples over time. For example, ifno antibody, or a subclinically significant level of an antibody, thatspecifically binds a VLA-4 binding antibody is determined to be presentin a first sample from a subject and a clinically significant thresholdof antibodies that specifically bind a VLA-4 binding antibody isdetermined to be present in a second or subsequent sample from thesubject, it may indicate the onset of an immune response to the VLA-4binding antibody in the subject. Progression of an immune response to aVLA-4 binding antibody in a subject may be indicated by the presence ofa higher level of an antibody that specifically binds a VLA-4 bindingantibody in a second or subsequent sample from a subject compared to thelevel present in the initial or previous sample from the subject.Regression of an immune response to a VLA-4 binding antibody may beindicated by the presence of a lower level of an antibody thatspecifically binds a VLA-4 binding antibody in a second or subsequentsample from a subject compared to the level present in the initial orprevious sample from the subject.

In one aspect of the invention, an immune response may be categorized aseither positive or negative based on whether a level of antibodiesagainst a therapeutic VLA-4 binding antibody is above a predeterminedclinically significant threshold. In some embodiments, a clinicallysignificant threshold is more than 1.645 (e.g. more than 2, 3, 4, 5, or6) standard deviations above a mean level of binding activity measuredin pre-immune subjects (i.e., subjects who have not received any dose oftherapeutic VLA-4 antibody). In some embodiments, the subjects arehealthy subjects and in certain embodiments, the subjects are diseasedpatients (e.g., patients with multiple sclerosis, Crohn's disease, orrheumatoid arthritis). In one embodiment, the threshold level is >0.5micrograms/ml serum. In some embodiments, the threshold level is equalto about 0.5 micrograms/ml serum.

In one embodiment, subjects who have been administered a VLA-4 bindingantibody may be categorized as falling into one of at least threecategories. One category, referred to herein as “negative”, includessubjects in which binding activities are at or below clinicallysignificant threshold levels (e.g., a subject in which antibodies to aVLA-4 binding antibody are not detected at a concentration of at leastabout 500 ng/ml in a biological sample (e.g., serum) obtained from thesubject). A second category, referred to herein as “transient positive”includes subjects in which binding activities are detected above athreshold level only a limited number of times, e.g., at one, two,three, four , five points in time (e.g., antibodies to a VLA-4 bindingantibody are not detected at a subsequent time point separated by atleast 30 days from the last time point). A third category, referred toherein as “persistent positive” includes subjects in which bindingactivities are detected above a threshold level a predetermined numberof times, e.g., at two, three, four, five, six, seven or more timepoints separated by a minimum threshold time interval (e.g., a subjectin which antibodies to a VLA-4 binding antibody are detected at aconcentration of at least about 500 ng/ml in two three, four, five, six,seven or more biological samples obtained from the subject at timepoints separated by at least a threshold interval). In some embodiments,the threshold interval is at least about 10, 20, 30, 40, 50, 60, 70, 80,90, or more days. Patients who are “persistent positive” may have a lossof efficacy from VLA-4 antibody therapy, while “transient positive”patients have full efficacy restored after only a temporary diminutionin efficacy.

It should be appreciated that a negative subject may become a transientpositive. And either one may become a persistent positive if a positiveimmune response develops and persists for a specified number of timepoints, e.g., at least two time points.

In one embodiment, a therapy may be changed based on a determination ofa single positive result. In another embodiment, a therapy may bechanged based upon a determination that a subject is a transientpositive subject. In yet another embodiment, a therapy may be changedbased upon a determination that a subject is a persistent positivesubject.

As discussed above, it should be appreciated that the terms transientand persistent are relative terms, and that a patient that seems to bepersistently positive may become negative at a later time. Accordingly,patients with positive responses should be monitored regularly toevaluate the persistence of the positive response, the effectiveness ofthe therapy, and/or the presence of other clinical manifestations of apositive immune response.

According to the invention, the risk of a reduction in therapeuticefficacy (e.g., the risk of a relapse) increases with the length of timethat a positive immune response persists. Accordingly, in one aspect ofthe invention, the number of times that a patient tests positive is lessimportant than the length of time over which the patient remainspositive. In one embodiment, a patient may be identified as being atrisk of a reduction in therapeutic efficacy (e.g., at risk of a relapse)if a positive result is detected within 3 months of the firstadministration of a therapeutic VLA-4 binding antibody. In oneembodiment, this risk increases if the positive immune response persistsfor 3-6 months, and further increases if the positive immune responsepersists for 6-9 months, and yet further increases with persistence for9-12 months after the first administration of the therapeutic VLA-4binding antibody. It should be appreciated that persistence for morethan one year even further increases the probability of a relapse.Accordingly, different therapeutic regimens may be appropriate for apatient with a persistently positive immune response. However, it shouldbe appreciated that even in the presence of a persistently positiveimmune response, a therapeutic antibody therapy need not be discontinuedunless it becomes ineffective (e.g., a loss of substantially allefficacy) or causes other negative clinical manifestations.

In some embodiments, treatment with a therapeutic VLA-4 binding antibodymay be discontinued if the treatment is ineffective or is losing iseffectiveness in a patient that has a below-threshold level of immuneresponse. A lack of efficacy (or a reduction in efficacy) in the absenceof a clinically significant immune response may indicate that theineffectiveness of the treatment is due to one or more factors otherthan a patient immune response to the therapeutic agent. A patient willnot be a transient positive if no positive response is detected.Accordingly, alternative treatment may be considered.

It should be appreciated that binding activities or antibody levels maybe compared to pre-immune activities or levels (i.e., measured beforethe administration of the first dose of VLA-4 binding antibody).However, a comparison to a pre-immune amount is not necessary asdiscussed herein, because a positive immune response may be identifiedwhen a clinically significant threshold (or above threshold) amount ofbinding activity or antibody levels are present in a patient sample.

Assays and Detection Methods

According to aspects of the invention, a threshold amount of an antibodyresponse is assayed for. As discussed herein, a qualitative assay may beperformed. Alternatively, a quantitative assay may be performed and inone embodiment, the quantitative data may be translated into aqualitative output (e.g., whether the amount of antibody is greater thana threshold amount).

Any suitable method for detecting an amount of antibody or bindingactivity may be used to determine whether it is at least a thresholdamount or activity. Detection assays may include any knownimmunodetection methods for detecting, confirming, binding, purifying,removing, quantifying and/or otherwise generally detecting antibodiesthat specifically bind to a specified therapeutic protein, e.g., to aVLA-4 binding antibody, or fragments thereof. For example,immunodetection techniques may include, but are not limited to, enzymelinked immunosorbent assays (ELISA) (including, but not limited to, astandard sandwich ELISA or a bridging ELISA), radioimmunoassays (RIA),immunoradiometric assays, fluoroimmunoassays, chemiluminescent assays,bioluminescent assays, radioimmunoprecipitation assays (RIPA), andWestern blots. In addition, immunodetection techniques may includeOptical Sensor-based methods, such as surface plasmon resonance (SPR),or guided mode resonance filter (BIND). Although certain examplesprovided herein relate to assays utilizing an immobilized VLA-4 bindingantibody attached to a surface (e.g. an ELISA), one of ordinary skill inthe art will recognize that the invention in some aspects may includeassays without surface attachment of a VLA-4 binding antibody (e.g. flowcytometric assays, etc.). Other immunodetection techniques may includeimmunoradiometric assays (IRMA), time-resolved fluorometry (TRF), orelectrochemiluminescence (ECL). A number of useful immunodetectionmethods have been described in the scientific literature, such as, e.g.,Doolittle M H and Ben-Zeev 0, 1999; Gulbis B and Galand P, 1993; DeJager R et al., 1993; and Nakamura et al., 1987, each incorporatedherein by reference.

In one aspect, an assay is performed to detect a presence of a bindingactivity for a VLA-4 binding antibody in a biological sample. In oneembodiment, the specificity of the binding activity may be evaluated bydetermining whether the observed binding activity is specific for theVLA-4 binding antibody or whether it is due to an interfering factorthat may be present in the biological sample such as a rheumatoid factoror other binding factor.

Aspects of the invention may include an assay that involves contacting abiological sample with an immobilization moiety to immobilize anybinding activity that is present in the biological sample. Immobilizedbinding activity may be detected using a detection moiety.Immobilization and detection moieties may be, respectively, immobilizedunlabeled and non-immobilized labeled VLA-4 binding antibodies asdescribed herein. In one embodiment, an immobilization moiety may bebound to a solid substrate or surface (e.g., in a well of a multi-wellplate, on the surface of an ELISA plate, etc.). In another embodiment,an immobilization moiety may be attached to a bead (e.g., a magneticbead) via a covalent or other linkage (e.g., the immobilization moietymay be conjugated to a biotin molecule and attached to a bead coatedwith streptavidin via a biotin-streptavidin interaction). In someembodiments, the bead may be attached to a surface or a matrix. Forexample, a magnetic bead may be immobilized on a magnetic surface.Similarly, a charged bead may be immobilized on a charged surface (e.g.,an electrode).

A positive result may be determined if the detected amount of bindingactivity (e.g., the amount of binding activity that is captured by theimmobilization moiety) is above a predetermined threshold. Thespecificity of the detected binding activity may be evaluated byincluding a competition moiety in the assay. The competition moiety maybe a non-immobilized unlabeled VLA-4 binding antibody that may beincluded to compete with the immobilization and/or detection steps ofthe assay. If the presence of the competition moiety reduces the bindingactivity by at least a predetermined percentage or cut-off, the bindingactivity is determined to be specific and the positive result isconfirmed. If the presence of the competition moiety fails to reduce thebinding activity by at least a predetermined percentage or cut-off, thebinding activity is determined to be non-specific and the initialpositive result is now determined to be a negative result for an immuneresponse.

According to aspects of the invention, predetermined amounts ofimmobilization, detection and/or competition moieties may be used.Similarly, an initial threshold level of binding activity may beestablished using a sample that contains a predetermined amount of anantibody that is known to bind to a VLA-4 binding antibody. For example,a threshold level may be established using between 10 ng and 1,000 ng(e.g., about 50 ng, or about 500 ng) of a control antibody per ml ofassay. The amount of antibody used to determine the threshold level willdetermine the sensitivity of the assay. In general, the sensitivity ofthe assay may be considered to be similar to the amount of antibody thatis used to determine the initial threshold. It should be appreciatedthat the amount of binding in the control may serve as a reference thatis used to determine the threshold (e.g., the threshold amount may be amultiple or a fraction of the signal obtained in the control). However,in one embodiment, the signal obtained in the control assay is used asthe threshold amount. It also should be appreciated that the assaysensitivity may be affected by a number of factors including theaffinity and specificity of the control antibody.

In one embodiment, the specificity of the binding activity may beevaluated by spiking the assay with an amount of competition moiety thatis similar to the amount of control antibody that was used to establishthe threshold level of binding. For example, an assay may be spiked withbetween about 10 ng and 1,000 ng (e.g., about 50 ng, or about 500 ng) ofunlabeled soluble VLA-4 binding antibody per ml of assay. However, otheramounts of competition moiety may be used.

Accordingly, in some embodiments of the invention, a first level ofbinding activity, in a biological sample, to the VLA-4 binding antibodyis determined in a first immunoassay for a first aliquot of thebiological sample, and a second level of binding activity to the VLA-4binding antibody is determined in a second immunoassay for a secondaliquot of the biological sample, wherein the second immunoassay isspiked with a greater amount of unlabeled soluble VLA-4 binding antibodythan the first immunoassay, and the presence of at least a thresholdlevel of soluble antibody to the VLA-4 binding antibody is indicated ifthe first level of binding activity is greater than a reference leveland the second level of binding activity is less than a predeterminedpercentage of the first level of binding activity.

One of ordinary skill in the art will recognize that different methodscan be used for assessing and/or monitoring an immune response in asubject who has been treated with a therapeutic antibody such as a VLA-4binding antibody. For example, as described above, the assessment and/ormonitoring may be performed by determining whether the amount of anantibody that specifically binds to a VLA-4 binding antibody using asingle-level “cut-off”. As used herein the cut-off level of binding isthe level at or above which increased detection will be scored assignificant and/or positive and a confirmatory determination that thedetection of a level of soluble binding activity in a biological samplereflects the level of an antibody that specifically binds to a VLA-4binding antibody in the sample. In other embodiments, the identificationof an immune response to the therapeutic VLA-4 binding antibody may beperformed quantitatively to determine a titer of an antibody thatspecifically binds to a VLA-4 binding antibody in a biological samplefrom a subject.

In one aspect of the invention, an immunodetection assay may be anELISA. As will be understood by those of ordinary skill in the art, theterm “ELISA” encompasses a number of protocols for immunodetection. Forexample, ELISA methods include sandwich ELISAs, bridging ELISAs, etc. Insome embodiments of the invention, the ELISA immunoassay is a manualassay. However, in some embodiments of the invention all or part of theELISA may be performed robotically.

In some embodiments of the invention, an ELISA assay includes using aVLA-4 binding antibody as an immobilized target moiety with which anELISA plate is coated. The coated ELISA plate may then be contacted witha biological sample for determination of the level of a subject antibodythat specifically binds a VLA-4 binding antibody. In some embodiments ofthe invention, a first aliquot of a biological sample is assayed usingan ELISA assay to determine the presence or absence of a thresholdamount of binding to the immobilized target VLA-4 binding antibody and asecond aliquot of the same biological sample is also assayed using anELISA to confirm whether or not a threshold (or above threshold) levelof binding to the immobilized target VLA-4 binding antibody isindicative of a VLA-4 binding antibody-specific antibody. In one aspectof the invention, the threshold level of soluble binding activity in analiquot is about equal to the level of binding activity present in acontrol or reference sample comprising at least about 50 ng, 100 ng, 200ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1,000 ngper ml of a reference antibody that binds to a VLA-4 binding antibody.In one embodiment, the threshold level is determined as about equal tothe level of binding activity in a control or reference samplecontaining about 500 ng/ml of a reference antibody that binds to a VLA-4binding antibody. The reference antibody may be polyclonal ormonoclonal. The reference antibody may be a murine anti-natalizumabantibody (e.g., 12C4 described in Sheremata et al., 1999, Neurology 52,page 1072). As described herein, if the level of binding to theimmobilized target VLA-4 binding antibody that is at least at thethreshold level, and the soluble binding activity is determined to bethe binding activity of an antibody that specifically binds to a VLA-4binding antibody, then it identifies an immune response to the VLA-4binding antibody in the subject.

In general, ELISA methods useful in methods of the invention may includeobtaining a biological sample from a subject who has been administered atherapeutic antibody such as a VLA-4 binding antibody (e.g. natalizumab,etc.) and contacting an aliquot of the sample with an immobilizationantibody. In some embodiments, the immobilization antibody may be thesame VLA-4 binding antibody that was administered to the subject. Theimmobilization antibody captures molecules or compounds in the samplethat bind to the antibody, and the sample is contacted with a seconddetection moiety that is capable of selectively binding to or detectingthe molecule or compound that is captured, (e.g., a labeled secondantibody). Examples of moieties capable of selectively binding ordetecting the complex include, but are not limited to antibodies orother ligands that can be labeled using a variety of markers (e.g.,biotin/avidin ligand binding arrangement, as is known in the art). Oneskilled in the art may also use a labeled third antibody. In preferredembodiments, the second moiety is a labeled form of the immobilizationantibody.

In some embodiments of the invention, an ELISA assay includes using thetherapeutic VLA-4 binding antibody as an immobilized target moiety withwhich an ELISA plate is coated. The coated ELISA plate may then becontacted with a biological sample for determination of the level of asubject antibody that specifically binds the therapeutic VLA-4 bindingantibody. In some embodiments of the invention, a first aliquot of abiological sample is assayed using an ELISA assay to determine thepresence or absence of a threshold amount of binding activity for theimmobilized target VLA-4 binding antibody, and a second aliquot of thesame biological sample is assayed using an ELISA to confirm whether ornot a threshold (or above threshold) level of binding to the immobilizedtarget VLA-4 binding antibody is indicative of a VLA-4 bindingantibody-specific antibody. In one aspect of the invention, thethreshold level of soluble binding activity in an aliquot is about equalto the level of binding activity present in a control or referencesample comprising at least about 50 ng, 100 ng, 200 ng, 300 ng, 400 ng,500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1,000 ng per ml of areference antibody that binds to a VLA-4 binding antibody. In oneembodiment, the threshold level is determined as about equal to thelevel of binding activity in a control or reference sample containingabout 500 ng/ml a reference antibody that binds to a VLA-4 bindingantibody. The reference antibody may be polyclonal or monoclonal. Thereference antibody may be a murine anti-natalizumab antibody (e.g., 12C4described in Sheremata et al., 1999, Neurology 52, page 1072).

A reference antibody that binds to a VLA-4 binding antibody may be areference antibody that binds to natalizumab (for example, an antibodythat binds to natalizumab with high affinity, e.g., with nanomolaraffinity). In some embodiments, a reference antibody that binds tonatalizumab may block natalizumab binding to VLA-4 (e.g., it may inhibitbinding of natalizumab to VLA-4 by at least 50%, at least 60%, at leasts70%, at least 80%, at least 90%, or more). The reference antibody may bea murine monoclonal antibody. In some embodiments, the referenceantibody is the 12C4 antibody (available from Maine BiotechnologyServices, Inc., Portland Me.; see, e.g., Sheremata et al., 1999,Neurology 52, page 1072). 12C4 is a blocking antibody that blocksnatalizumab binding to VLA-4. In some embodiments, the referenceantibody competes with 12C4 for binding to natalizumab. Antibody bindingcompetition may be demonstrated using standard methods of assessing anantibody's ability to competitively inhibit the 12C4 antibody's abilityto block binding of natalizumab to VLA-4. In some embodiments, thepresence of an antibody that specifically binds to a VLA-4 bindingantibody is determined using a bridging ELISA. In a bridging ELISA,antibodies that specifically bind to a VLA-4 binding antibody (e.g.,from a biological sample) act as a bridge between VLA-4 binding antibodycoated on an ELISA plate and detectably labeled VLA-4 binding antibodyin solution (e.g., non-immobilized). Thus, an ELISA signal afterstandard processing indicates that the detectable label has been linkedto the solid phase and that a soluble binding activity is present in thebiological sample.

Contacting an aliquot of the biological sample with the immobilizedantibody under effective conditions and for a period of time sufficientto allow the formation of immune complexes (primary immune complexes) isgenerally a matter of adding the aliquot of the biological sample to theimmobilized antibody (e.g., a VLA-4 binding antibody immobilized on anELISA plate) and incubating the mixture for a period of time long enoughfor the immobilized antibody to form an immune complex with (i.e., tobind to) a molecule or compound with soluble binding activity that ispresent in the aliquot of the biological sample. The molecule orcompound with soluble binding activity may be an induced antibody thatspecifically binds to the VLA-4 binding antibody or may be a non-inducedendogenous antibody or receptor that binds to the VLA-4 binding antibody(e.g., a rheumatoid factor [RF] or an anti-Fab antibody). After thistime, the sample-antibody mixture (e.g., the ELISA plate, dot blot, orwestern blot) will generally be washed to remove unbound antibodyspecies and/or materials from the assay mixture.

If a threshold level of binding activity is detected, an additional stepmay involve confirming whether or not the binding activity is indicativeof an induced antibody that specifically binds to the therapeutic VLA-4binding antibody. For this confirmation step, a second aliquot of thebiological sample may be prepared and assayed as described for the firstaliquot, except that a predetermined amount of non-immobilized unlabeledcompetition VLA-4 binding antibody also is added to the assay (e.g., theELISA assay). For example, the predetermined amount of competitionantibody may be an unlabeled amount that reduces a specific signal byabout 50% or more in a control assay. If the presence of the unlabeledVLA-4 binding antibody reduces the signal by more than an expectedpercentage amount, then the threshold (or above threshold) bindingactivity is judged as a positive indicator for the presence of anantibody that specifically binds to the therapeutic VLA-4 bindingantibody. In contrast, if the presence of the unlabeled VLA-4 bindingantibody reduces the signal by less than an expected percentage amountthen the threshold (or above threshold) binding activity is judged asnegative for an antibody that specifically binds to a VLA-4 bindingantibody. It should be appreciated that a non-specific signal may be dueserum factors other than an antibody that binds to the VLA-4 bindingantibody. As used herein the terms “spike” or “spiked” refers to theaddition of an unlabeled (or differently labeled) soluble competitionVLA-4 binding antibody to a sample or assay.

As used herein, the “percentage reduction” is the percentage of thelevel of binding determined in the first aliquot. Thus, for example, ifthere is an indication of about 500 ng/ml equivalent of a molecule orcompound with binding activity in the first aliquot and the inclusion ofthe unlabeled VLA-4 binding antibody in the second aliquot reduces theamount of signal by more than 40-90% (e.g., by about 50% or more , byabout 55% or more, by about 60% or more, by about 65% or more, by about70% or more, by about 75% or more, by about 80% or more, by about 85% ormore, by about 90% or more), then the binding activity in the biologicalsample is considered indicative of the presence of an induced antibodythat specifically binds to the therapeutic VLA-4 binding antibody. Butif the inclusion of the unlabeled VLA-4 binding antibody in the secondaliquot reduces the amount of signal by less than 20-40% , then thebinding activity in the biological sample is not considered to beindicative of the presence of an induced antibody that specificallybinds to the therapeutic VLA-4 binding antibody. In some embodiments,the competition antibody may be soluble unlabeled natalizumab. In someembodiments, the soluble unlabeled natalizumab may be used at a finalconcentration of about 100 μg/ml. However, any concentration of freeunlabeled natalizumab may be used if it results in a predetermineddecrease (e.g., about 40%, about 50%, or more) in the signal obtainedfor a control sample containing a control amount of reference antibody.For example, a control sample may contain about 500 ng/ml, about 3 μg/mlor any other suitable amount of reference antibody (e.g., 12C4). Asindicated herein, the presence in a biological sample from a patient ofan antibody that specifically binds to a VLA-4 binding antibodyindicates that the subject has a clinically significant immune responseto the VLA-4 binding antibody.

In an exemplary “sandwich” ELISA, a therapeutic VLA-4 binding antibody(e.g., natalizumab) may be used as the target antibody and may beimmobilized onto a selected surface exhibiting protein affinity, such asa well in a polystyrene microtiter plate. Then, a sample from a subjectwho has had at least one administration of a therapeutic VLA-4 bindingantibody, e.g., natalizumab, is added to the wells. After binding and/orwashing to remove non-bound materials, binding molecules or compoundsthat are bound to the target antibody may be detected. Detection may beachieved by the addition of a second antibody that is linked to adetectable label. In addition, the identity of the binding molecule orcompound as an antibody that specifically binds to a VLA-4 bindingantibody may be confirmed as described above herein.

As will be understood by those of ordinary skill in the art,notwithstanding individual features (e.g. the confirmatory stepsdescribed herein), in general, ELISAs have certain features in common,such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes.

In coating a plate with either antigen or antibody, the wells of theplate will generally be incubated with a solution of the targetantibody, either overnight or for a specified period of hours. A coatingbuffer may be a sodium phosphate/BSA coating buffer or another suitableart-known coating buffer. The wells of the plate will then be washed toremove incompletely adsorbed material. Any remaining available surfacesof the wells are then “coated” with a nonspecific protein that isantigenically neutral with regard to the test sample. This protein maybe bovine serum albumin (BSA), casein or solutions of milk powder, etc.The coating allows for blocking of nonspecific adsorption sites on theimmobilizing surface and thus reduces the background caused bynonspecific binding of antisera onto the surface.

In an ELISA, a secondary or tertiary detection means may be used or adirect detection means may be used. When using a secondary or tertiarydetection methods, after binding of a protein or antibody to the well,coating with a non-reactive material to reduce background, (e.g. withblocking buffer such as Tris-sucrose blocking buffer or otherart-recognized blocking buffer), and washing to remove unbound material,the immobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand. In preferred embodiments of theinvention the second binding ligand is a VLA-4 binding antibody (e.g.the same VLA-4 binding antibody as used for the target antibody).

As used herein, the term “under conditions effective to allow immunecomplex formation” means that the conditions preferably include dilutingthe antigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG), phosphate buffered saline (PBS)/Tween, PBS with caseinand Tween 20, or PBS/BSA buffer with Tween 20. Various other art-knownassay diluents can be used in the methods of the invention. These addedagents also tend to assist in the reduction of nonspecific backgroundand may include up to 0.5 M NaCl.

As used herein, the “suitable” conditions also mean that the incubationis at a temperature or for a period of time sufficient to alloweffective binding. Incubation steps are typically from about 1 to 2 to 4hours or so, at temperatures preferably on the order of 25° C. to 27°C., or may be overnight at about 4° C. Various art-known assaytemperature and timing parameters can be used in the methods of theinvention.

After the incubation steps in an ELISA, the contacted surface is washedto remove non-bound material. A preferred washing procedure may includewashing with a solution such as PBS/Tween, TBS/Tween, or borate buffer,which may also include up to 0.5 M NaCl. Following the formation ofspecific immune complexes between the test sample and the targetantibody, and subsequent washing, the presence of even minute amounts ofimmune complexes may be determined. It will be understood thatadditional art-known wash buffer formulations can be used in the methodsof the invention.

To provide a detecting means, the second or third antibody will have anassociated detectable label. In certain embodiments, the detectablelabel is an enzyme that will generate color development upon incubatingwith an appropriate chromogenic substrate. Thus, for example, one maycontact or incubate the first and second immune complex with a urease-,glucose oxidase-, alkaline phosphatase-, hydrogen peroxidase-conjugatedantibody, or other conjugated antibody for a period of time and underconditions that favor the development of further immune complexformation (e.g., incubation for 2 hours at room temperature in aPBS-containing solution such as PBS-Tween).

It also will be understood by those of skill in the art that one or morepositive and negative quality controls may be utilized in the methods ofthe invention. A positive quality control sample may be a normal serumsample that contains a predetermined amount of an antibody that is knownto bind to a VLA-4 binding antibody. Quality control samples may bereacted in parallel with and under the same conditions as the biologicaland control samples of the assay and provide a measure of the functionof the assay. A negative quality control sample may be a serum sampleknown not to include an antibody that is known to bind to a VLA-4binding antibody One of ordinary skill will understand how to utilizepositive and negative control reactions and samples in an ELISA toascertain and validate the functionality of the solutions and/orsubstrates and/or protocol used in the assay. For example, a positivecontrol may include a known amount of an antibody that specificallybinds the VLA-4 binding antibody so when treated under the sameconditions as the test samples (e.g., the biological sample) itindicates that the assay works within expected parameters. An example ofa negative control may be a sample that is known to not include anantibody that specifically binds to the VLA-4 binding antibody. Such anegative control, when treated under the same conditions as the testsample (e.g., the biological sample), will demonstrate that the bindingdetected in a biological sample arises from the biological sample and isnot due to contamination of assay components or other factor notassociated with the biological sample. A non-limiting example of anassay encompassed by the methods of the invention may involve thefollowing procedures. Coating the wells of an ELISA plate with asolution of about 0.25 μg/mL natalizumab reference standard in a bufferand incubating the coated plate overnight at ambient temperature;washing the plate wells a least once with a wash buffer and incubatingthe plate wells with a blocking buffer for a minimum of 1 hour atambient temperature; diluting control samples and screening samples byabout 1:10 in an assay diluent; diluting competition samples andnatalizumab (at about 1 mg/mL) together in the assay diluent to a finalconcentration of natalizumab of about 100 μg/mL and about a 1:10 finaldilution of the competition samples; incubating control samples,screening samples, and competition samples about 1 hour at ambienttemperature and washing the plate wells at least once with wash buffer;incubating the samples in the plate wells between about 60 and 150minutes at ambient temperature and washing the plate wells at leastthree times with wash buffer; adding about 100 μL/plate well ofbiotinylated-natalizumab diluted to about 1:1000 in the assay diluent,incubating the plate for about 60-90 min at ambient temperature, andwashing the plate wells at least three times with wash buffer; addingstreptavidin-horseradish peroxidase diluted about 1:5000 in assaydiluent to the samples in plate wells, incubating the plate about 60-90minutes at ambient temperature, and washing the plate at least threetimes with wash buffer; adding a sufficient amount of color-producingsubstrate to the plate wells to visualize antibody binding, developingthe plate for several minutes at ambient temperature, stopping thedevelopment by adding 1N H2SO4 to the plate wells; and reading the platewells thereby obtaining a result.

It also is contemplated that the ELISA reagents described herein maybepackaged in a kit that may be produced commercially to detect thepresence of and/or measure an antibody that specifically binds a VLA-4binding antibody in a biological sample as described herein.

It will also be understood that the controls for use in the inventionmay include, in addition to predetermined values (such as clinicallysignificant threshold values identified as described herein for aparticular therapeutic protein), samples of materials tested in parallelwith the experimental materials. Examples include negative controlsamples (e.g., generated through manufacture) to be tested in parallelwith the experimental samples.

As used herein, a biological sample may be, but is not limited to, anyof the following: a body fluid of a subject including, blood, serum,plasma, urine, saliva, pleural effusions, stool, synovial fluid,cerebral spinal fluid, mucus, and tissue infiltrations. Preferred bodyfluids include blood, plasma, and serum. As used herein, biologicalsamples may be obtained using methods well known to those of ordinaryskill in the related medical arts. A biological sample may be obtaineddirectly from a subject or may be obtained from cell, tissue, or otherculture. A biological sample may be fresh or may have been stored undersuitable conditions (e.g., frozen, chilled, etc.) prior to use inmethods of the invention.

It should be appreciated that a biological sample may be obtained from asubject at different time intervals after the administration of atherapeutic agent. However, a therapeutic agent may have acharacteristic in vivo half-life, and the amount of free therapeuticagent that is present in a biological sample typically will decrease asa function of time after administration of the agent to the subject. Thepresence of free therapeutic agent (unlabeled and non-immobilized) in abiological sample may interfere with binding and detection reactions ofthe invention. Accordingly, biological samples should be obtained aslong as possible after a therapeutic administration, for example at a“trough” in the treatment cycle. A “trough” represents the lowestamounts of free therapeutic agent that are present in a subject during atreatment cycle. A trough may occur, for example, long after onetherapeutic administration and soon before a subsequent therapeuticadministration. It should be appreciated that the timing of the troughmay be influenced by many factors, including the amount of agent that isadministered, the half-life of the agent, the frequency ofadministration, etc. For example, a monthly administration of a VLA-4binding antibody (e.g., 300 mg of natalizumab) results in a trough atabout 30 days after one administration and immediately before asubsequent administration. However, it should be appreciated that assaysof the invention may be performed on samples taken at different timesafter treatment administration provided that the assays are relativelyinsensitive to, or account for, the presence of free therapeutic agentin the biological sample. When levels of binding activity are comparedfor different samples taken at different time points, it may beparticularly important to obtain each sample from a similar stage in thetreatment cycle (e.g., a similar length of time after a therapeuticadministration) so that the results can be interpreted without needingto correct for differences in levels of free therapeutic agent in thebiological samples.

It should be appreciated that biological samples may be diluted beforebeing assayed (e.g., 2 fold, 5 fold, 10 fold, 50 fold, 100 fold, andincluding higher or lower fold values or any fold value in between). Inone embodiment, a reference sample containing a clinically significantthreshold amount of reference antibody may be diluted by the same amountas the biological sample being tested so that the signal obtained forthe biological sample can be compared directly to the signal obtainedfor the reference sample.

In some embodiments of the invention, one or more aliquots from abiological sample are used. As used herein, the term “aliquot” means aportion or part of the biological sample. In some embodiments, two ormore aliquots may be taken from a biological sample obtained from asubject and the aliquots can be tested using methods of the invention todetermine the presence of an immune response to a VLA-4 binding antibodyin the subject. For example, two substantially equivalent aliquots canbe taken from a biological sample obtained from a subject to whom aVLA-4 binding antibody (e.g. natalizumab) has been administered, and alevel of soluble binding activity can be detected in one aliquot (e.g. a“first” aliquot) can be determined. Additionally, the other aliquot(e.g. the “second” aliquot) may be assessed using the methods of theinvention to determine whether the soluble binding activity detected inthe first aliquot, which would also be present in the second aliquot byvirtue of their common sample origin, is an antibody that specificallybinds a VLA-4 binding antibody. In some embodiments, if at least athreshold level of binding is present in the first aliquot and thesoluble binding activity is determined to be the activity of an antibodythat specifically binds a VLA-4 binding antibody, it identifies that animmune response to the VLA-4 binding antibody is present in the subject.

VLA-4 Binding Antibodies

Aspects of the invention relate to detection assay(s) for identifyingand/or monitoring an immune response to one or more therapeutic VLA-4binding antibodies that are administered to a subject. In certainaspects of the invention, a detection assay involves both immobilizationand detection moieties. In some embodiments, a detection assay also mayinclude a competition moiety. As described herein, an immobilizationmoiety may be used to immobilize an induced antibody (e.g., a serumantibody) that binds to the VLA-4 binding antibody. A detection moietymay be used to detect the immobilized antibody. A competition moiety maybe used to compete with the either the immobilization and/or detectionmoiety for binding to the induced antibody in order to determine thespecificity of binding to the induced antibody. The immobilization anddetection moieties may bind independently to one or more antibodies thatare characteristic of an immune response to a VLA-4 binding antibody.Accordingly, the immobilization and detection moieties may be the sameVLA-4 binding antibody that was administered to the subject (e.g., theimmobilization moiety may be an immobilized form of the therapeuticVLA-4 binding antibody that was administered to the subject, and thedetection moiety may be a labeled form of the therapeutic VLA-4 bindingantibody that was administered to the subject). However, in otherembodiments, each of the immobilization and detection moietiesindependently may be variants of the therapeutic VLA-4 binding antibodythat was administered to the subject, provided that the immobilizationand detection moieties bind with suitable affinity to detect one or moreinduced antibodies (e.g., serum antibodies) against the therapeuticVLA-4 binding antibody. The competition moiety is typically a solubleunlabeled (or differentially labeled) form of the immobilization ordetection moiety. However, the competition moiety may be a variant ofthe immobilization or detection moiety, provided that it competessufficiently for binding to the therapeutic VLA-4 binding antibody todetermine the specificity of the binding activity detected in the assay.

According to aspects of the invention, any one or more VLA-4 bindingantibodies (including natalizumab and/or related VLA-4 bindingantibodies) may be used therapeutically. Accordingly, any one or more ofthe VLA-4 binding antibodies may be used as immobilization, detection,and/or competition moieties in a detection assay of the invention. Inone embodiment, a VLA-4 binding antibody may be an IgG antibody (e.g.,an IgG4 antibody). In another embodiment, a VLA-4 binding antibody maybe polyclonal or monoclonal. In yet another embodiment, a VLA-4 bindingantibody may be a humanized version of a murine antibody.

Natalizumab and related VLA-4 binding antibodies are described, e.g., inU.S. Pat. No. 5,840,299. mAb 21.6 and HP1/2 are exemplary murinemonoclonal antibodies that bind VLA-4. Natalizumab is a humanizedversion of murine mAb 21.6 (see, e.g., U.S. Pat. No. 5,840,299). Ahumanized version of HP1/2 has also been described (see, e.g., U.S. Pat.No. 6,602,503). Several additional VLA-4 binding monoclonal antibodies,such as HP2/1, HP2/4, L25 and P4C2, are described (e.g., in U.S. Pat.No. 6,602,503; Sanchez-Madrid et al., 1986 Eur. J. Immunol.,16:1343-1349; Hemler et al., 1987 J. Biol. Chem. 2:11478-11485; Issekutzand Wykretowicz, 1991, J. Immunol., 147: 109 (TA-2 mab); Pulido et al.,1991 J. Biol. Chem., 266(16):10241-10245; and U.S. Pat. No. 5,888,507).Many useful VLA-4 binding antibodies interact with VLA-4 on cells, e.g.,lymphocytes, but do not cause cell aggregation. However, otheranti-VLA-4 binding antibodies have been observed to cause suchaggregation. HP1/2 does not cause cell aggregation. The HP1/2 MAb(Sanchez-Madrid et al., 1986 Eur. J. Immunol., 16:1343-1349) has anextremely high potency, blocks VLA-4 interaction with both VCAM1 andfibronectin, and has the specificity for epitope B on VLA-4. Thisantibody and other B epitope-specific antibodies (such as B1 or B2epitope binding antibodies; Pulido et al., 1991 J. Biol. Chem.,266(16):10241-10245) represent one class of useful VLA-4 bindingantibodies.

An exemplary VLA-4 binding antibody has one or more CDRs, e.g., allthree HC CDRs and/or all three LC CDRs of a particular antibodydisclosed herein, or CDRs that are, in sum, at least 80, 85, 90, 92, 94,95, 96, 97, 98, or 99% identical to such an antibody, e.g., natalizumab.In one embodiment, the H1 and H2 hypervariable loops have the samecanonical structure as those of an antibody described herein. In oneembodiment, the L1 and L2 hypervariable loops have the same canonicalstructure as those of an antibody described herein.

In one embodiment, the amino acid sequence of the HC and/or LC variabledomain sequence is at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100%identical to the amino acid sequence of the HC and/or LC variable domainof an antibody described herein, e.g., natalizumab. The amino acidsequence of the HC and/or LC variable domain sequence can differ by atleast one amino acid, but no more than ten, eight, six, five, four,three, or two amino acids from the corresponding sequence of an antibodydescribed herein, e.g., natalizumab. For example, the differences may beprimarily or entirely in the framework regions.

The amino acid sequences of the HC and LC variable domain sequences canbe encoded by a sequence that hybridizes under high stringencyconditions to a nucleic acid sequence described herein or one thatencodes a variable domain or to a nucleic acid encoding an amino acidsequence described herein. In one embodiment, the amino acid sequencesof one or more framework regions (e.g., FR1, FR2, FR3, and/or FR4) ofthe HC and/or LC variable domain are at least 70, 80, 85, 90, 92, 95,97, 98, 99, or 100% identical to corresponding framework regions of theHC and LC variable domains of an antibody described herein. In oneembodiment, one or more heavy or light chain framework regions (e.g., HCFR1, FR2, and FR3) are at least 70, 80, 85, 90, 95, 96, 97, 98, or 100%identical to the sequence of corresponding framework regions from ahuman germline antibody.

Calculations of “homology” or “sequence identity” between two sequences(the terms are used interchangeably herein) are performed as follows.The sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). The optimal alignment isdetermined as the best score using the GAP program in the GCG softwarepackage with a Blossum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences.

The skilled artisan will realize that conservative amino acidsubstitutions may be made in VLA-4 binding antibodies to providefunctionally equivalent variants, of the antibodies, i.e, the variantsretain the functional capabilities of the VLA-4 polypeptides. As usedherein, a “conservative amino acid substitution” refers to an amino acidsubstitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references that compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Exemplary functionally equivalentvariants of VLA-4 binding antibodies include conservative amino acidsubstitutions of in the amino acid sequences of proteins disclosedherein. Conservative substitutions of amino acids include substitutionsmade amongst amino acids within the following groups: (a) M, I, L, V;(b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

As used herein, the term “hybridizes under high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. High stringency hybridizationconditions include hybridization in 6× SSC at about 45° C., followed byone or more washes in 0.2× SSC, 0.1% SDS at 65° C., or substantiallysimilar conditions.

Antibodies can be tested for a functional property, e.g., VLA-4 binding,e.g., as described in U.S. Pat. No. 6,602,503.

Antibody Generation

Antibodies that bind to VLA-4 can be generated by immunization, e.g.,using an animal. All or part of VLA-4 can be used as an immunogen. Forexample, the extracellular region of the alpha-4 subunit can be used asimmunogen. In one embodiment, the immunized animal containsimmunoglobulin producing cells with natural, human, or partially humanimmunoglobulin loci. In one embodiment, the non-human animal includes atleast a part of a human immunoglobulin gene. For example, it is possibleto engineer mouse strains deficient in mouse antibody production withlarge fragments of the human Ig loci. Using the hybridoma technology,antigen-specific monoclonal antibodies derived from the genes with thedesired specificity may be produced and selected. See, e.g., XenoMouse™,Green et al. Nature Genetics 7:13-21 (1994), US Published PatentApplication No. 2003-0070185, U.S. Pat. No. 5,789,650, and WO 96/34096.Non-human antibodies to VLA-4 can also be produced, e.g., in a rodent.The non-human antibody can be humanized, e.g., as described in U.S. Pat.No. 6,602,503, EP 239 400, U.S. Pat. No. 5,693,761, and U.S. Pat. No.6,407,213.

EP 239 400 (Winter et al.) describes altering antibodies by substitution(within a given variable region) of their complementarity determiningregions (CDRs) for one species with those from another. CDR-substitutedantibodies are predicted to be less likely to elicit an immune responsein humans compared to true chimeric antibodies because theCDR-substituted antibodies contain considerably less non-humancomponents. (Riechmann et al., 1988, Nature 332, 323-327; Verhoeyen etal., 1988, Science 239, 1534-1536). Typically, CDRs of a murine antibodysubstituted into the corresponding regions in a human antibody by usingrecombinant nucleic acid technology to produce sequences encoding thedesired substituted antibody. Human constant region gene segments of thedesired isotype (usually gamma I for CH and kappa for CL) can be addedand the humanized heavy and light chain genes are co-expressed inmammalian cells to produce soluble humanized antibody.

Queen et al., 1989 Proc Natl Acad Sci USA. Dec; 86(24):10029-33 and WO90/07861 have described a process that includes choosing human Vframework regions by computer analysis for optimal protein sequencehomology to the V region framework of the original murine antibody, andmodeling the tertiary structure of the murine V region to visualizeframework amino acid residues which are likely to interact with themurine CDRs. These murine amino acid residues are then superimposed onthe homologous human framework. See also U.S. Pat. Nos. 5,693,762;5,693,761; 5,585,089; and 5,530,101. Tempest et al., 1991, Biotechnology9, 266-271) utilize, as standard, the V region frameworks derived fromNEWM and REI heavy and light chains respectively for CDR-graftingwithout radical introduction of mouse residues. An advantage of usingthe Tempest et al., approach to construct NEWM and REI based humanizedantibodies is that the three-dimensional structures of NEWM and REIvariable regions are known from x-ray crystallography and thus specificinteractions between CDRs and V region framework residues can bemodeled.

Non-human antibodies can be modified to include substitutions thatinsert human immunoglobulin sequences, e.g., consensus human amino acidresidues at particular positions, e.g., at one or more of the followingpositions (preferably at least five, ten, twelve, or all): (in the FR ofthe variable domain of the light chain) 4L, 35L, 36L, 38L, 43L, 44L,58L, 46L, 62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L,87L, 98L, and/or (in the FR of the variable domain of the heavy chain)2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H,73H, 74H, 75H, 78H, 91H, 92H, 93H, and/or 103H (according to the Kabatnumbering). See, e.g., U.S. Pat. No. 6,407,213.

As will be apparent to one of ordinary skill in the art, the presentinvention also provides for F(ab′)2, Fab, Fv and Fd fragments; chimericantibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/orlight chain CDR3 regions have been replaced by homologous human ornon-human sequences; chimeric F(ab′)2 fragment antibodies in which theFR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences.

In certain embodiments, a VLA-4 binding antibody may be a VLA-4single-chain antibody, a single-domain antibody, or a Nanobody™.Characteristics of each of these antibody types and methods for theiruse are well known in the art. Nanobodies™ are the smallest functionalfragments of antibodies and are derived from naturally occurringsingle-chain antibodies (see Ablynx, Belgium; ablynx.com). Nanobody™technology was developed following the discovery that camelidae (camelsand llamas) possess a unique repertoire of fully functional antibodiesthat lack light chains. Nanobody™ structure consists of a singlevariable domain (VHH), a hinge region, and two constant domains (CH2 andCH3). The cloned and isolated VHH domain is a stable polypeptideharboring the full antigen-binding capacity of the original heavy chain.Nanobodies™ combine the features of conventional antibodies withfeatures of small molecule drugs. Nanobodies™ show high targetspecificity and low inherent toxicity. Additionally, Nanobodies™ arevery stable, can be administered by means other than injection, and areeasy to manufacture. In certain embodiments, a therapeutic VLA-4 bindingantibody, an immobilization moiety, and/or a detection moiety may be ahumanized Nanobody™.

Antibody Production

Fully human monoclonal antibodies that bind to VLA-4 can be produced,e.g., using in vitro-primed human splenocytes, as described by Boerneret al., 1991, J. Immunol., 147, 86-95. They may be prepared byrepertoire cloning as described by Persson et al., 1991, Proc. Nat.Acad. Sci. USA, 88: 2432-2436 or by Huang and Stollar, 1991, J. Immunol.Methods 141, 227-236. U.S. Pat. No. 5,798,230. Large nonimmunized humanphage display libraries may also be used to isolate high affinityantibodies that can be developed as human therapeutics using standardphage technology (see, e.g., Vaughan et al, 1996 Nat Biotechnol. Mar;14(3):309-14; Hoogenboom et al. (1998) Immunotechnology 4:1-20; andHoogenboom et al. (2000) Immunol Today 2:371-8; US Published PatentApplication No. 2003-0232333). Antibodies can be produced in prokaryoticand eukaryotic cells. In one embodiment, the antibodies (e.g., scFvs)are expressed in a yeast cell such as Pichia (see, e.g., Powers et al.(2001) J Immunol Methods. 251:123-35), Hanseula, or Saccharomyces. Inone embodiment, antibodies, particularly full length antibodies, e.g.,IgGs, are produced in mammalian cells. Exemplary mammalian host cellsfor recombinant expression include Chinese Hamster Ovary (CHO cells)(including dhfr- CHO cells, described in Urlaub and Chasin (1980) Proc.Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker,e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621),lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, COScells, K562, and a cell from a transgenic animal, e.g., a transgenicmammal. For example, the cell is a mammary epithelial cell.

In addition to the nucleic acid sequence encoding the immunoglobulindomain, the recombinant expression vectors may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017). Exemplary selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr- hostcells with methotrexate selection/amplification) and the neo gene (forG418 selection).

In an exemplary system for recombinant expression of an antibody (e.g.,a full length antibody or an antigen-binding portion thereof), arecombinant expression vector encoding both the antibody heavy chain andthe antibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to enhancer/promoter regulatory elements (e.g., derived fromSV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLPpromoter regulatory element or an SV40 enhancer/AdMLP promoterregulatory element) to drive high levels of transcription of the genes.The recombinant expression vector also carries a DHFR gene, which allowsfor selection of CHO cells that have been transfected with the vectorusing methotrexate selection/amplification. The selected transformanthost cells are cultured to allow for expression of the antibody heavyand light chains and intact antibody is recovered from the culturemedium. Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells, and recover the antibody from theculture medium. For example, some antibodies can be isolated by affinitychromatography with a Protein A or Protein G. U.S. Pat. No. 6,602,503also describes exemplary methods for expressing and purifying a VLA-4binding antibody.

Antibodies may also include modifications, e.g., modifications thatalter Fc function, e.g., to decrease or remove interaction with an Fcreceptor or with C1q, or both. For example, the human IgG1 constantregion can be mutated at one or more residues, e.g., one or more ofresidues 234 and 237, e.g., according to the numbering in U.S. Pat. No.5,648,260. Other exemplary modifications include those described in U.S.Pat. No. 5,648,260.

For some antibodies that include an Fc domain, the antibody productionsystem may be designed to synthesize antibodies in which the Fc regionis glycosylated. For example, the Fc domain of IgG molecules isglycosylated at asparagine 297 in the CH2 domain. This asparagine is thesite for modification with biantennary-type oligosaccharides. Thisglycosylation participates in effector functions mediated by Fcyreceptors and complement C1q (Burton and Woof (1992) Adv. Immunol.51:1-84; Jefferis et al. (1998) Immunol. Rev. 163:59-76). The Fc domaincan be produced in a mammalian expression system that appropriatelyglycosylates the residue corresponding to asparagine 297. The Fc domaincan also include other eukaryotic post-translational modifications.

Antibodies can also be produced by a transgenic animal. For example,U.S. Pat. No. 5,849,992 describes a method for expressing an antibody inthe mammary gland of a transgenic mammal. A transgene is constructedthat includes a milk-specific promoter and nucleic acids encoding theantibody of interest and a signal sequence for secretion. The milkproduced by females of such transgenic mammals includes,secreted-therein, the antibody of interest. The antibody can be purifiedfrom the milk, or for some applications, used directly.

As used herein, the VLA-4 binding antibodies of the invention may besubstantially full length VLA-4 binding antibodies or functionalfragments thereof. For example, if a fragment of a VLA-4 bindingantibody is sufficient to allow specific binding by an antibody thatspecifically binds a VLA-4 binding antibody it is a functional VLA-4binding antibody and may be used in the methods and kits of theinvention. One of ordinary skill in the art will be able to identifyVLA-4 binding antibody fragments and determine whether a VLA-4 bindingantibody fragment is a functional VLA-4 binding antibody fragment usingonly routine procedures and binding assays. Thus, descriptions andexamples of methods of using immobilized and non-immobilized VLA-4binding antibodies that are provided herein, also apply to the use offunctional immobilized and non-immobilized VLA-4 binding antibodyfragments.

Labels and Detection

Aspects of the invention include using non-immobilized, anti-therapeuticprotein antibodies (e.g., VLA-4 binding antibodies) as detectionmoieties to assess the presence and/or level of soluble binding activitythat is bound to an immobilized antibody against a therapeutic protein(e.g., a target VLA-4 binding antibody). Methods to evaluate thepresence and/or level of soluble binding activity may include the use ofone or more labeled detection moieties (e.g., a VLA-4 binding antibodycontaining or attached to a detectable label). A detectable label isdefined as any moiety that can be detected using an assay. Theantibodies and functional antibody fragments of the invention can becoupled to specific labeling agents for detecting binding according tostandard coupling procedures. A wide variety of detectable labels can beused, such as those that provide direct detection (e.g., a radioactivelabel, a fluorophore, [e.g. Green Fluorescent Protein (GFP), RedFluorescent Protein (RFP), etc.], a chromophore, an optical or electrondense label, etc.) or indirect detection (e.g., an enzyme tag such ashorseradish peroxidase, etc.). Non-limiting examples of detectablelabels that have been attached to or incorporated into antibodiesinclude: enzymes, radiolabels, fluorescent labels, phosphorescentmolecules, chemiluminescent molecules, chromophores, luminescentmolecules, photoaffinity molecules, and colored particles or ligandssuch as biotin, etc. In addition, detection methods of the invention mayinclude electrochemiluminescence methods (ECL).

A variety of methods may be used to detect a label, depending on thenature of the label and other assay components. Labels may be directlydetected through optical or electron density, radioactive emissions,non-radiative energy transfers, etc. or indirectly detected withantibody conjugates, strepavidin-biotin conjugates, etc. Many additionaldetectable labels are known in the art, as are methods for theirattachment to antibodies.

Labeled antibodies of the invention may be antibodies that are used invitro, e.g., in an immunoassay such as an ELISA. Such detectably labeledantibodies may be antibodies that have a detectable label incorporatedinto the antibody or may be antibodies that are linked to a secondarybinding ligand and/or to an enzyme (an enzyme tag) that will generate acolored product upon contact with a chromogenic substrate. Examples ofsuitable enzymes include urease, alkaline phosphatase, (horseradish)hydrogen peroxidase or glucose oxidase. Preferred secondary bindingligands are biotin and/or avidin and streptavidin compounds. The use ofsuch labels is well known to those of skill in the art and aredescribed, for example, in U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; eachincorporated herein by reference.

Numerous methods for the attachment or conjugation of an antibody to itsdetectable label are known in the art. An attachment method may includethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3 .alpha.-6.alpha.-diphenylglycouril-3 attached tothe antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporatedherein by reference). Monoclonal antibodies may also be reacted with anenzyme in the presence of a coupling agent such as glutaraldehyde orperiodate. Antibodies may be labeled with fluorescein markers in thepresence of these coupling agents or by reaction with an isothiocyanate.In other embodiments, antibodies may be labeled by derivatization, forexample, by selectively introducing sulfhydryl groups in the Fc regionof the antibody, using reaction conditions that do not alter theantibody recognition site.

The detection of the detectable label in an assay of the invention isalso referred to herein as detecting the “signal”. Methods for detectingthe signal in an immunoassay are well known in the art. In someimportant embodiments of the invention, the assay signal can be detectedusing with a multi-well plate reader (e.g. microplate reader) to assessthe amount and/or location of a signal. Signal detection can be opticaldetection or other detection means suitable for detecting a detectablelabel utilized in the invention. Additional methods for detecting labelsare well known in the art and can be used in methods of the invention.Methods of the invention include ELISAs that have a sensitivity fordetecting an antibody that specifically binds to a VLA-4 bindingantibody, wherein the sensitivity is at least about 1000 ng, 500 ng, or50 ng. In preferred embodiments, the ELISA sensitivity for detecting anantibody that specifically binds to a VLA-4 binding antibody is at leastabout 500 ng.

In general, the detection of immunocomplex formation may be achievedthrough the application of numerous approaches. These methods aregenerally based upon the detection of a label or marker, such as any ofthose radioactive, fluorescent, biological and enzymatic tags. U.S.Patents concerning the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241, each incorporated herein by reference. Of course, one mayfind additional advantages through the use of a secondary binding ligandsuch as a second antibody and/or a biotin/avidin ligand bindingarrangement, as is known in the art.

It should be appreciated that techniques described herein to obtain andproduce a VLA-4 binding antibody (e.g., natalizumab) also may be used toobtain and produce a reference antibody that binds to the VLA-4 bindingantibody with high affinity (for example, a high affinity natalizumabbinding antibody (e.g., 12C4).

Kits

The invention also relates, in part, to kits for assaying the presenceof an immune response to a VLA-4 binding antibody in a sample. Anexample of such a kit may include the above-mentioned antibodiesincluding, but not limited to Natalizumab or other VLA-4 bindingantibody, or a fragment thereof. A kit may include detectably labeledand/or unlabeled antibodies and may include solutions and compounds fordetectably labeling an antibody. A kit of the invention may also includeone or more of the following components: plates, pipettes, vials,detectable label, solutions (e.g. blocking buffer, wash buffer, bindingsolutions, diluent solutions, etc), positive and/or negative controlsamples and solutions. A kit of the invention may also include writteninstructions for the use of the kit for the identification of an immuneresponse to a VLA-4 binding antibody in a biological sample. Kits of theinvention may also include additional components useful in theperformance of positive and/or negative ELISA control assays. A kit ofthe invention may also include equipment such as plate readers and/orrobotic instrumentation for use in the methods of the invention.

EXAMPLES Example 1 Natalizumab Immunogenicity Assay

The assay format and design was based upon a bridging enzyme-linkedImmunosorbent assay (ELISA). Standard reagents and procedures suitablefor bridging assays were used. In brief, using standard proceduresnatalizumab was adsorbed to the surface of microtiter plates followed bya blocking step to minimize non-specific binding of serum antibodies.Controls and samples were diluted and added to the wells. Using standardprocedures, detection of bound anti-natalizumab antibodies wasaccomplished by incubating the plates with biotin-natalizumab followedby streptavidin-Alkaline Phosphatase (SA-AP). The color development wasproportional to the amount of anti-natalizumab antibody bound.

Natalizumab was diluted to a specific concentration in plate coatingbuffer was incubated in 96-well microtiter ELISA plates overnight atambient temperatures. The recommended volume was 100 μl/well. The plateswere washed with washing buffer then incubated with blocking buffer atambient temperatures for >1 hr. The volume of blocking buffer was 200μl/well. The plates were washed with washing buffer and then incubatedwith quality control and subject (patient) samples diluted 1:10 in assaydiluent with or without added natizumab (100 μg/ml final concentration)at ambient temperatures for 2 hr±15 min. The assay diluent containingadded natalizumab represented a confirmatory step. The volume for theassay diluent was 100 μl/well. The plates were then washed in washingbuffer, and then incubated with SA-AP at ambient temperatures for 30-35min. The volume for the incubation with SA-AP was 100 μl/well. Theplates were then washed in washing buffer, and then incubated with APsubstrate, p-nitrophenyl phosphate (PNPP) at ambient temperatures for45-50 min. The volume for the incubation in the AP substrate was 100μl/well. Stop solution (e.g. H5O4, etc.) was then added directly to thesubstrate reaction mixture and the optical density (OC) was read at 405nm.

For assay acceptance the positive and negative controls had to provideresults within a pre-defined value and precision. For the assessment ofsubject (patient) samples results, the samples were judged as negativeif their OD value fell below the defined negative cut-off qualitycontrol sample. Samples were also judged negative if their OD value fellabove the defined negative cut-off quality control sample and theinhibition of the subject (patient) sample signal by added natalizumabin the confirmatory step was less than a pre-defined percent. Sampleswere judged positive if their OD value fell above the defined negativecut-off quality control sample and the inhibition of the subject(patient) sample signal by added natalizumab in the confirmatory stepwas greater than or equal to a pre-defined percent.

Example 2 Screening Assay for an Immune Response to a VLA-4 Antibody

A screening assay (as described above) was performed on samples fromsubjects who had been administered natalizumab. The presence of animmune response to a VLA-4 antibody was examined in patients who hadundergone natalizumab administration.

Biological samples from subjects who had been administered natalizumabwere tested for the presence of a soluble antibody that specificallybinds natalizumab using the methods described in Example 1.

The tests included the use of a high affinity mAb as a positive control.The high affinity mAb detected binding antibodies (e.g. antibodies thatbound natalizumab) that were present in biological samples fromsubjects. The negative cut-off level was determined to the lowestconcentration that returned acceptable accuracy/precision (25%/25%); 50ng/mL in 10% serum. The sensitivity of the mAb was 500 ng/mL in neat(undiluted) serum. With respect to sensitivity, the mAb was determinedto detect anti- natalizumab Abs but to be insensitive to irrelevanthuman monoclonal antibodies. The interference with the mAb by othermolecules or components of the samples was determined to includeinterference by free drug at an mAb:drug ratio of greater than 1:2. FIG.1 illustrates the effects of free natalizumab interference with theimmunoassay for anti-natalizumab antibodies. Rheumatoid factor was alsofound to interfere with the assay.

In addition to the screening assay described in Examples 1 and 2,characterization assays to assess binding in the screening assays werealso preformed. Biological samples from subjects who had beenadministered natalizumab were tested for the presence of a solubleantibody that specifically binds natalizumab using a flow cytometryblocking assay. A high affinity mAb was used as a positive control. Theassay detected blocking antibodies. The negative cut-off level for themAb used was determined at 3-4 standard deviations above the mean ofmeasured levels in sera obtained from non-dosed patients. There was a 5%false-positive rate and the sensitivity of the assay was 500 ng/ml ofneat serum. The specificity of the mAb was tested and the mAb wasdetermined to detect anti- natalizumab antibodies but was found to beinsensitive to irrelevant human monoclonal antibodies. Free-druginterference with the assay was also examined. The assay showed thepresence of free-drug interference. Results from an assessment ofblocking assay interference by free drug are shown in FIG. 2.

Blocking Assay for Natalizumab Results

The positive results of the screening and blocking assays were compared.There were 217 Blocking assay positives and 222 Screening assaypositives, thus there was a difference of 5% and a percent concordanceof 98%. Accordingly, the screening assay provides an accurate measure ofa subject's immune response to a therapeutic VLA-4 binding antibody.

Example 3

A screening assay (as described in Examples 1 and 2) was performed onsamples from 625 subjects who had been administered natalizumab. Thepresence of an immune response to a VLA-4 antibody was examined inpatients who had undergone natalizumab administration. Incidence ofantibodies to natalizumab in the patients examined was: 91% (569patients) antibody negative and 9% (56 patients) “binding antibody”positive at any time point with 3% (19 patients) “transiently” positiveand 6% (37 patients) “persistently” positive. The transient positivepatients had detectable antibodies (at a concentration of >0.5 μg/ml) ata single time point, but negative for antibodies at all other timepoints. The persistent positive patients had detectable antibodies attwo or more time points that were at least 42 days apart, or at a singletime point with no follow-up samples tested.

FIG. 3 shows the time of first positive results in patients whodeveloped any antibodies. Six percent of patients developed “persistent”antibodies to natalizumab. Over 90% of persistent-positive patientsfirst had detectable antibodies at week 12. No subject became positivefor persistent antibodies after week 36. Transient-positive patients haddetectable antibodies at week 12, but were subsequently antibodynegative.

The results were analyzed to determine the presence of an effect ofantibodies on the rate of relapse of the original disorder in thepatients treated. FIG. 4 illustrates the overall effect of antibodies onrelapse rate. The relapse rate was also assessed for subjects withrespect to the time elapsed from the administration of the natalizumab.FIG. 5A-D depict the rate of relapse at 0-3 months, 3-6 months, 6-9months, and 9-12 months respectively.

Results

The results indicated that there is no apparent effect of antibodiesduring the first three months of treatment. From three to six months the“transient” antibody-positive patients showed diminution in efficacy ofthe natalizumab treatment. “Persistent” antibody-positive patientsshowed lost of efficiency of natalizumab treatment. From six to twelvemonths, full efficacy was restored in “transient” antibody-positivepatients, but not in “persistent” antibody-positive patients.Accordingly it is important to identify transient antibody-positivepatients as a target population for continued VLA-4 binding antibodytherapy.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

1-71. (canceled)
 72. A method, comprising: (a) administering natalizumab to a subject having Multiple Sclerosis; (b) detecting in a first biological sample obtained from the subject at a first time point the presence or absence of at least a threshold level of anti-natalizumab antibody binding activity; and (c) detecting in a second biological sample obtained from the subject at a second time point the presence or absence of at least a threshold level of anti-natalizumab antibody binding activity, wherein the threshold level is equal to the level of anti-natalizumab antibody binding activity present in a reference sample comprising at least 500 ng/ml of anti-natalizumab antibody.
 73. The method of claim 72, wherein the first time point is at least 12 weeks following step (a).
 74. The method of claim 72, wherein the second time point is at least 42 days following the first time point.
 75. The method of claim 72 further comprising identifying the subject for continued treatment with natalizumab if: (i) the anti-natalizumab antibody binding activity detected in step (b) is present at or above the threshold level and if the anti-natalizumab antibody binding activity detected in step (c) is absent or present below the threshold level; or (ii) the anti-natalizumab antibody binding activity detected in step (b) is absent or present below the threshold level and if the anti-natalizumab antibody binding activity detected in step (c) is absent or present below the threshold level.
 76. The method of claim 72 further comprising identifying the subject for modified or discontinued treatment with natalizumab if the anti-natalizumab antibody binding activity detected in step (b) is present at or above the threshold level and if the anti-natalizumab antibody binding activity detected in step (c) is present at or above the threshold level.
 77. The method of claim 72, wherein the level of anti-natalizumab antibody binding activity in step (b) and/or in step (c) is detected using a bridging enzyme-linked immunosorbent assay (ELISA) assay.
 78. A method, comprising: (a) administering natalizumab to a subject having Multiple Sclerosis; (b) detecting in a first serum sample obtained from the subject at a first time point of at least 12 weeks following the administration of natalizumab, the presence or absence of at least a threshold level of anti-natalizumab antibody binding activity; and (c) detecting in a second serum sample obtained from the subject at a second time point of at least 42 days following the first time point, the presence or absence of at least a threshold level of anti-natalizumab antibody binding activity, wherein the threshold level is equal to the level of anti-natalizumab antibody binding activity present in a reference sample comprising at least 500 ng/ml of anti-natalizumab antibody.
 79. The method of claim 78 further comprising identifying the subject for continued treatment with natalizumab if: (i) the anti-natalizumab antibody binding activity detected in step (b) is present at or above the threshold level and if the anti-natalizumab antibody binding activity detected in step (c) is absent or present below the threshold level; or (ii) the anti-natalizumab antibody binding activity detected in step (b) is absent or present below the threshold level and if the anti-natalizumab antibody binding activity detected in step (c) is absent or present below the threshold level.
 80. The method of claim 78 further comprising identifying the subject for modified or discontinued treatment with natalizumab if the anti-natalizumab antibody binding activity detected in step (b) is present at or above the threshold level and if the anti-natalizumab antibody binding activity detected in step (c) is present at or above the threshold level.
 81. The method of claim 79, wherein the level of anti-natalizumab antibody binding activity in step (b) and/or in step (c) is detected using a bridging ELISA assay.
 82. The method of claim 80, wherein the level of anti-natalizumab antibody binding activity in step (b) and/or in step (c) is detected using a bridging ELISA assay.
 83. A method, comprising: (a) obtaining at a first time point a first biological sample from a subject that has been administered natalizumab; (b) detecting in the first biological sample the presence or absence of at least a threshold level of anti-natalizumab antibody binding activity; (c) obtaining at a second time point a second biological sample from the subject; and (d) detecting in the second biological sample the presence or absence of at least a threshold level of anti-natalizumab antibody binding activity, wherein the threshold level of step (b) and step (d) is equal to the level of anti-natalizumab antibody binding activity present in a reference sample comprising at least 500 ng/ml of anti-natalizumab antibody.
 84. The method of claim 83, wherein the first time point is at least 12 weeks following step (a).
 85. The method of claim 83, wherein the second time point is at least 42 days following the first time point.
 86. The method of claim 83 further comprising identifying the subject for continued treatment with natalizumab if: (i) the anti-natalizumab antibody binding activity detected in step (b) is present at or above the threshold level and if the anti-natalizumab antibody binding activity detected in step (c) is absent or present below the threshold level; or (ii) the anti-natalizumab antibody binding activity detected in step (b) is absent or present below the threshold level and if the anti-natalizumab antibody binding activity detected in step (c) is absent or present below the threshold level.
 87. The method of claim 83 further comprising identifying the subject for modified or discontinued treatment with natalizumab if the anti-natalizumab antibody binding activity detected in step (b) is present at or above the threshold level and if the anti-natalizumab antibody binding activity detected in step (c) is present at or above the threshold level.
 88. The method of claim 83, wherein the level of anti-natalizumab antibody binding activity in step (b) and/or in step (c) is detected using a bridging enzyme-linked immunosorbent assay (ELISA) assay.
 89. A method, comprising: (a) obtaining at a first time point a first serum sample from a subject having Multiple Sclerosis that has been administered natalizumab, wherein the first time point is at least 12 weeks following administration of natalizumab; (b) detecting in the first serum sample the presence or absence of at least a threshold level of anti-natalizumab antibody binding activity, wherein the threshold level of step (b) is equal to the level of anti-natalizumab antibody binding activity present in a reference sample comprising at least 500 ng/ml of anti-natalizumab antibody; (c) obtaining at a second time point a second serum sample from the subject, wherein the second time point is at least 42 days following the first time point; and (d) detecting in the second serum sample the presence or absence of at least a threshold level of anti-natalizumab antibody binding activity, wherein the threshold level of step step (d) is equal to the level of anti-natalizumab antibody binding activity present in a reference sample comprising at least 500 ng/ml of anti-natalizumab antibody.
 90. The method of claim 89 further comprising identifying the subject for continued treatment with natalizumab if: (i) the anti-natalizumab antibody binding activity detected in step (b) is present at or above the threshold level and if the anti-natalizumab antibody binding activity detected in step (d) is absent or present below the threshold level; or (ii) the anti-natalizumab antibody binding activity detected in step (b) is absent or present below the threshold level and if the anti-natalizumab antibody binding activity detected in step (d) is absent or present below the threshold level.
 91. The method of claim 89 further comprising identifying the subject for modified or discontinued treatment with natalizumab if the anti-natalizumab antibody binding activity detected in step (b) is present at or above the threshold level and if the anti-natalizumab antibody binding activity detected in step (d) is present at or above the threshold level.
 92. The method of claim 90, wherein the level of anti-natalizumab antibody binding activity in step (b) and/or in step (d) is detected using a bridging ELISA assay.
 93. The method of claim 91, wherein the level of anti-natalizumab antibody binding activity in step (b) and/or in step (c) is detected using a bridging ELISA assay. 